CN111471606A

CN111471606A - 一株优化的高产岩藻糖基乳糖的酿酒酵母菌株及其应用

Info

Publication number: CN111471606A
Application number: CN202010187632.9A
Authority: CN
Inventors: 刘巍峰; 孟祥锋; 徐明远
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2020-07-31
Anticipated expiration: 2040-03-17
Also published as: CN111471606B

Abstract

本发明公开了一株优化的高产2’‑岩藻糖基乳糖的酿酒酵母工程菌株，通过代谢工程改造，使得酿酒酵母可以吸收乳糖并在胞内以GDP‑甘露糖为前体合成GDP‑岩藻糖，并通过异源表达来源于蜡样芽孢杆菌中的α‑1,2‑岩藻糖基转移酶，所得到的重组菌株可以高产2’‑岩藻糖基乳糖，进而通过后续过表达GDP‑甘露糖合成相关酶将2’‑岩藻糖基乳糖产量提升至约3.8g/L，这为酿酒酵母工业化生产2’‑岩藻糖基乳糖提供了重要的参考。

Description

一株优化的高产岩藻糖基乳糖的酿酒酵母菌株及其应用

技术领域

本发明属于基因工程领域，尤其涉及一株高产岩藻糖基乳糖，尤其是高产2’-岩藻糖基乳糖的重组酿酒酵母及其应用。

背景技术

2’-岩藻糖基乳糖(2-FL)作为母乳寡糖中的重要组成部分，可以促进人体肠道内有益微生物如双歧杆菌等的生长，降低肠道内病原体感染的风险，还可以调控免疫应答。目前2-FL已在婴幼儿配方奶粉中作为添加剂使用。2-FL的大量合成具有良好的经济效益。化学合成2-FL过程中需添加有毒试剂，难以应用于食品工业。

在生物体内合成2-FL是行之有效的方法。2-FL的生物合成过程已较为清楚：一分子供体GDP-岩藻糖在α-1,2-岩藻糖基转移酶的作用下与受体乳糖结合，产生一分子2-FL。现有技术中有研究构建了高产2-FL的大肠杆菌重组菌株。但是大肠杆菌可能产生内毒素，影响产品的分离纯化难度及产品安全性；另外大肠杆菌发酵时易受到噬菌体的污染，这使大肠杆菌工业化生产2-FL受到了制约。

酿酒酵母是被普遍认为安全的微生物，已有研究利用酿酒酵母合成2-FL；但是，现有报道中酿酒酵母菌株合成的2-FL产量仅0.5g/L，难以满足工业化生产的需要。制约酿酒酵母中2-FL产量的重要因素之一为α-1,2-岩藻糖基转移酶的酶活性不足。寻找具有高活力的α-1,2-岩藻糖基转移酶可能会提高酿酒酵母中2-FL的产量。

发明内容

本发明通过代谢工程改造，使得酿酒酵母可以吸收乳糖并在胞内以GDP-甘露糖为前体合成GDP-岩藻糖，并通过异源表达来源于蜡样芽孢杆菌中的α-1,2-岩藻糖基转移酶，得到了高产2-FL的酿酒酵母工程菌株。

一方面，本发明提供了一株高产2’-岩藻糖基乳糖的酿酒酵母工程菌株，所述酿酒酵母工程菌株中含有重组的乳糖透性酶(Lactosepermease)、重组的GDP-甘露糖脱水酶(GDP-mannose-4,6-dehydratase)、重组的GDP-岩藻糖合酶(GDP-L-fucose synthase)以及重组的α-1,2-岩藻糖基转移酶(α-1,2fucosyltransferase)。

本发明中，术语“重组”指通过转化将目的基因转移到酿酒酵母中；所述含有重组的目的基因的酿酒酵母工程菌株与其来源的野生型酿酒酵母相比，其已经以遗传方式修饰，从而展示出改变的或不同的基因型和/或表型。可以通过将目的蛋白通过载体在宿主菌中进行表达(包括过表达的方式)来实现，还可以通过将目的蛋白的编码基因重组到宿主菌的基因组上来实现。

含有重组的目的基因的酿酒酵母工程菌株含有至少一个被引入的基因序列，此类基因序列包括但不限于天然存在或不存在酿酒酵母中的基因以及希望引入到酿酒酵母细胞中的其他基因或DNA序列，无论野生型的酿酒酵母中是否已经存在相同或类似的基因或DNA序列；在一些实施方式中，引入的基因序列将通过例如同源重组或定点诱变来修饰或甚至替代内源基因或DNA序列；在其他的实施方式中，引入到酿酒酵母工程菌株中的重组基因可以是与存在酿酒酵母中的DNA序列相同的并且被引入以提供DNA的一个或多个另外的拷贝，从而DNA的基因产物过表达或修饰的表达。

在优选的实施方式中，所述α-1,2-岩藻糖基转移酶来源于蜡样芽孢杆菌(Bacillus cereus)，更优选的，所述α-1,2-岩藻糖基转移酶的氨基酸序列如SEQ ID No.11所示。

更优选的，所述酿酒酵母工程菌株包含重组的α-1,2-岩藻糖基转移酶的编码基因；优选的，所述编码基因的序列如SEQ ID No.12所示。

优选的，所述乳糖透性酶来源于乳酸克鲁维斯酵母，更优选的，所述乳糖透性酶的氨基酸序列如SEQ ID No.1所示。

优选的，所述GDP-甘露糖脱水酶和GDP-岩藻糖合酶来源于大肠杆菌，优选，来源于大肠杆菌K12，更优选的，所述GDP-甘露糖脱水酶的氨基酸序列如SEQ ID No.3所示，所述GDP-岩藻糖合酶的氨基酸序列如SEQ ID No.5所示。

进一步的，所述酿酒酵母工程菌株还包括重组的甘露糖磷酸异构酶(Phosphomannomutase)、重组的GDP-甘露糖焦磷酸酶(GDP-mannose pyrophosphorylase)。

优选的，所述甘露糖磷酸异构酶和GDP-甘露糖焦磷酸酶来源于酿酒酵母，所述甘露糖磷酸异构酶的氨基酸序列如SEQ ID No.19所示；所述GDP-甘露糖焦磷酸酶的氨基酸序列如SEQ ID No.21所示。

进一步的，所述酿酒酵母工程菌株还包括重组的甘露糖-6-磷酸异构酶(Mannose-6-phosphate isomerase)；优选的，所述甘露糖-6-磷酸异构酶来源于酿酒酵母，其氨基酸序列如SEQ ID No.23所示。

进一步的，所述酿酒酵母工程菌株还包括无线粒体信号肽NADH激酶(NADHkinase)；优选的，所述无线粒体信号肽NADH激酶来源于酿酒酵母，其氨基酸序列如SEQ ID No.25所示。

在优选的实施方式中，所述酿酒酵母工程菌株的出发菌株为酿酒酵母W303-1a。

另一方面，本发明还提供了所述的酿酒酵母工程菌株的制备方法，所述方法包括将所述目的基因重组到酿酒酵母中的步骤。

优选的，将所述目的基因整合到酿酒酵母的基因组中。

进一步的，所述目的基因的整合位点包括TRP1、URA3、LEU2或HIS3。

另一方面，本发明还提供了所述酿酒酵母工程菌株在生产2’-岩藻糖基乳糖中的应用。

本发明以酿酒酵母W303-1a为出发菌株。从克鲁维斯酵母总DNA中克隆出乳糖透性酶Lac12，构建表达盒P_gal1-lac12-T_cyc1，并通过无缝融合的方法将表达盒与pRS304连接。所得到的重组载体以限制性内切酶Bsu36I线性化后，通过醋酸锂转化法转入酿酒酵母W303-1a中，并通过色氨酸缺陷的YNB固体平板筛选得到阳性克隆。进一步PCR验证后，所得到的正确转化子命名为FL01。FL01可以吸收胞外添加的乳糖。从大肠杆菌K12总DNA中克隆出GDP-甘露糖脱水酶(Gmd)以及GDP-岩藻糖合成酶(WcaG)，以来源于pUMRI-A的双向启动子gal1,10构建双向表达盒P_gal1-gmd-T_cyc1&P_gal10-wcaG-T_adh1。将以上表达盒通过无缝融合的方法与pRS306连接，所得到的重组载体以BstBI限制性内切酶线性化，随后通过醋酸锂转化法转入FL01中，验证正确的重组菌株命名为FL03，该菌株可以吸收胞外的乳糖，同时可以以胞内的GDP-甘露糖为底物，合成GDP-岩藻糖。

以包括Helicobacterpylori、Acetobacter sp.、Bacillus cereus、Bacteroidesunifomis、Bacteroides eggerthii、Neocallimastixcaliforniae等菌株来源的α-1,2-岩藻糖基转移酶为基础，以PCR融合的方法构建表达盒，将表达盒连接在载体pRS305上，并通过醋酸锂转化法分别转入FL03中，其中，表达来源于Bacillus cereus的α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株FL06可以产生约2.9g/L的2-FL。

随后针对酿酒酵母中合成2-FL的前体GDP-甘露糖的合成途径进行改造，以半乳糖诱导型启动子过表达Pmi40、Sec53和Psa1三个负责将果糖-6-磷酸转化为GDP-甘露糖的酶，将酿酒酵母重组菌株中2-FL产量提高至约3.7g/L。进一步通过过表达无线粒体信号肽的NADH激酶，将2-FL产量提高至3.8g/L。

现有技术中也有报道在酿酒酵母中合成2-FL，但是，所得到的酿酒酵母重组菌株中的2-FL产量仅为约0.5g/L。而本申请中利用来源于蜡样芽孢杆菌的α-1,2-岩藻糖基转移酶构建的酿酒酵母重组菌株，以半乳糖诱导相关基因的表达，发酵96h后2-FL产量可以达到2.9g/L，进而通过后续过表达GDP-甘露糖合成相关酶将2-FL产量提升至约3.8g/L，这为酿酒酵母工业化生产2-FL提供了重要的参考。

附图说明

图1.表达Lac12的酿酒酵母重组菌株FL01可以将乳糖吸收至胞内；A，葡萄糖标准品；B，半乳糖标准品；C，乳糖标准品；D、E，表达Lac12的酿酒酵母重组菌株S-L胞内裂解液。

图2.异源表达Gmd和WcaG的酿酒酵母重组菌株FL03可以产生GDP-岩藻糖；A，GDP-岩藻糖标准品；B，表达Lac12的酿酒酵母重组菌株FL01；C，表达乳糖转运体Lac12和GDP-岩藻糖合成相关酶WcaG和Gmd的酿酒酵母重组菌株FL03。

图3.重组菌株FL04菌株可以产生2-FL；a，酿酒酵母重组菌株FL03；b,酿酒酵母重组菌株FL04。

图4.表达不同来源α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株中2-FL产量；FL04，表达来源于Helicobacterpylori中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL05，表达来源于Acetobacter sp.中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL06，表达来源于Bacillus cereus中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL07，表达来源于Bacteroides eggerthii中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL08，表达来源于Bacteroides unifomis中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL09，表达来源于Neocallimastixcaliforniae中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株。以上菌株的出发菌株均为FL03。

图5.过表达甘露糖合成途径相关酶可以提高酿酒酵母FL06中2-FL的产量；FL06，表达来源于Bacillus cereus中α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株；FL10，在菌株FL06基础上表达甘露糖合成途径相关酶Psa1和Sec53的酿酒酵母重组菌株；FL11，在菌株FL06基础上表达甘露糖合成途径相关酶Psa1、Sec53和Pmi40的酿酒酵母重组菌株。

图6.菌株FL18中2-FL产量及产生能力进一步提高；A，FL18中2-FL产量提高；B，FL18中2-FL产生效率提高。

实施方式

下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。

实施例一、可吸收乳糖的重组酿酒酵母基因工程菌株的构建

本实施例中的重组工程菌株的构建，以酿酒酵母W303-1a为出发菌株。

利用Omega试剂盒提取乳酸克鲁维斯酵母(Kluyveromyceslactis)的总DNA，使用引物Lac12-F和Lac12-R从其中扩增出乳糖透性酶(Lactosepermease)的序列，命名为Lac12，其氨基酸序列如SEQ ID No.1所示，核酸序列如SEQ ID No.2所示。

以酿酒酵母(Saccharomyces cerevisiae)W303-1a总DNA为模板，以BamHI-GAL1-F和GAL1-LAC12-R为引物扩增出gal1启动子，以lac12-CYC1-F和CYC1-XhoI-R扩增出cyc1终止子，通过SOE-PCR构建P_gal1-lac12-T_cyc1表达盒，并通过无缝连接的方法连接在以BamHI和XhoI双酶切后的pRS304载体上，得到重组载体pRS304-P_gal1-lac12-T_cyc1。重组载体pRS304-P_gal1-lac12-T_cyc1以Bsu36I酶切，通过切胶纯化得到线性化片段。

以醋酸锂转化法，将线性化的pRS304-P_gal1-lac12-T_cyc1转入酿酒酵母W303-1a中，整合位点为TRP1。涂布于色氨酸缺陷的YNB固体平板上。转化子长出后挑取于色氨酸缺陷的YNB液体培养基中，提取总DNA进行PCR验证，正确的为阳性克隆子，命名为FL01。

上述引物序列如下表所示：

FL01菌株吸收乳糖能力验证：将FL01于添加2％半乳糖和0.2％乳糖的YP培养基中培养24h后，离心去上清，菌体冲洗两遍以后破碎，所得到的细胞裂解液进行TLC分析，TLC条件：薄层硅胶板购自Thermo公司，展开剂为水：冰醋酸：异丁醇＝1：1：2，10h后，60℃下将硅胶板吹干，随后将硅胶板置于染色液(90％甲醇，10％浓硫酸，0.2％苔黑素)中浸润染色，260℃下吹干，显色。可以检测到胞内存在乳糖，如图1所示。

实施例二、产GDP-岩藻糖的重组酿酒酵母基因工程菌株的构建

从大肠杆菌(Escherichia coli)K12中，以引物GMD-F和GMD-R扩增出GDP-甘露糖脱水酶(GDP-mannose-4,6-dehydratase)的序列，命名为Gmd，其氨基酸序列如SEQ ID No.3所示，其核苷酸序列如SEQ ID No.4所示。以引物WcaG-F和WcaG-R扩增出GDP-岩藻糖合酶(GDP-L-fucose synthase)的序列，命名为WcaG，其氨基酸序列如SEQ ID No.5所示，其核苷酸序列如SEQ ID No.6所示。

以pUMRI-A质粒(Ye,L.,X.Lv,and H.Yu,Assembly of biosynthetic pathwaysin Saccharomyces cerevisiae using a marker recyclable integrative plasmidtoolbox.Frontiers of Chemical Engineering in China,2017.11(1):p.126-132.)为模板，wcag-10-F和10-gmd-R为引物扩增出双向启动子gal1,10。以酿酒酵母W303-1a总DNA为模板，以SacI-ADH1-F和ADH1-wcaG-R引物扩增出adh1终止子序列，以gmd-CYC1-F和CYC1-KpnI-R引物扩增出cyc1终止子序列。通过SOE-PCR的方法，将以上片段依次连接，得到双向基因表达盒P_gal1-gmd-T_cyc1&P_gal10-wcaG-T_adh1。并通过无缝连接的方法连在SacI和KpnI酶切后的载体pRS306上，得到重组载体pRS306-P_gal1-gmd-T_cyc1&P_gal10-wcaG-T_adh1。重组载体pRS306-P_gal1-gmd-T_cyc1&P_gal10-wcaG-T_adh1以NcoI酶线性化，切胶回收。

将线性化后的pRS306-P_gal1-gmd-T_cyc1&P_gal10-wcaG-T_adh1通过醋酸锂转化法转入实施例1得到的菌株FL01中，整合位点为URA3，涂布于色氨酸、尿嘧啶缺陷的YNB固体平板上。转化子长出后挑取于色氨酸、尿嘧啶缺陷的YNB液体培养基中，提取总DNA以引物SacI-ADH1-F和CYC1-KpnI-R进行PCR验证，正确的为阳性克隆子，命名为FL03。

所用引物如下表所示：

FL03菌株中GDP-岩藻糖的检测：将FL03于添加2％葡萄糖和2％半乳糖的YP培养基中培养48h后，离心去上清，菌体冲洗两遍以后破碎，所得到的细胞裂解液进行HPLC分析。所用HPLC仪器来自岛津公司，带有UV检测器，使用C18分析柱(SyncronisaQ 250*4.6*5μm,Thermo Fisher Scientific)进行分析，柱温30℃，流动相为20mM三乙胺，添加2％(v/v)乙腈，流速为0.6mL/min，样品进样量15μL，检测波长为254nm；可以检测到胞内存在GDP-岩藻糖，检测结果如图2所示，表明FL03菌株可以产生GDP-岩藻糖。

实施例三、产2’-岩藻糖基乳糖的重组酿酒酵母基因工程菌株的构建

以人类及幽门螺杆菌来源的α-1,2-岩藻糖基转移酶为模板，通过blast分析从Helicobacterpylori、Acetobacter sp.、Bacillus cereus、Bacteroides unifomis、Bacteroides eggerthii、Neocallimastixcaliforniae菌株中发现新的α-1,2-岩藻糖基转移酶，分别命名为Futhp(氨基酸序列如SEQ ID No.7所示，核苷酸序列如SEQ ID No.8所示)，Futas(氨基酸序列如SEQ ID No.9所示，核苷酸序列如SEQ ID No.10所示)，Futbc(氨基酸序列如SEQ ID No.11所示，核苷酸序列如SEQ ID No.12所示)，Futbu(氨基酸序列如SEQ ID No.15所示，核苷酸序列如SEQ ID No.16所示)，Futbe(氨基酸序列如SEQ ID No.13所示，核苷酸序列如SEQ ID No.14所示)，Futnc(氨基酸序列如SEQ ID No.17所示，核苷酸序列如SEQ ID No.18所示)。

将上述α-1,2-岩藻糖基转移酶编码序列以如下表所示引物扩增。以酿酒酵母W303-1a总DNA为模板分别扩增得到对应以上α-1,2-岩藻糖基转移酶的启动子gal1和终止子cyc1。通过SOE-PCR的方法融合对应片段，得到不同α-1,2-岩藻糖基转移酶的表达盒P_gal1-fut-T_cyc1。将表达盒分别与XbaI和HindIII酶切后的pRS305无缝连接，得到重组载体。所用引物如下表所示：

以上得到的重组载体均以BspTI酶切后，切胶回收。

将线性化后的上述构建的重组pRS305-P_gal1-fut-T_cyc1载体通过醋酸锂转化法转入实施例2得到的菌株FL03中，整合位点为LEU2，涂布于色氨酸、尿嘧啶、亮氨酸缺陷的YNB固体平板上。转化子长出后挑取于色氨酸、尿嘧啶、亮氨酸缺陷的YNB液体培养基中，提取总DNA进行PCR验证，正确的为阳性克隆子，将带有来源于Helicobacterpylori、Acetobactersp.、Bacillus cereus、Bacteroides unifomis、Bacteroides eggerthii、Neocallimastixcaliforniae菌株的α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株分别命名为FL04，FL05，FL06，FL07，FL08，FL09。

上述重组菌株中2-FL的检测方法：将菌株于添加2％葡萄糖、2％半乳糖和0.2％乳糖的YP培养基中培养96h后，离心取上清为胞外产物，菌体冲洗两遍以后破碎，所得到的细胞裂解液为胞内产物。将胞外及胞内产物进行HPLC分析。所用HPLC仪器来自岛津公司，带有RI检测器，使用Rezex ROA-Organic Acid H+(8％)column(Phenomenex,Torrance,CA,USA)分析柱进行分析，柱温50℃，流动相为0.005N的H₂SO₄，流速为0.6mL/min，样品进样量15μL，检测结果如图3所示，以重组菌株FL04为例，可在保留时间7.9min处检测到2-FL。进一步对表达不同来源α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株所产生的2-FL进行定量分析，结果如图4所示，其中，表达来源于Bacillus cereus(蜡样芽孢杆菌)的α-1,2-岩藻糖基转移酶的酿酒酵母重组菌株FL06中2-FL产量可达到约2.9g/L。

实施例四、酿酒酵母重组菌株FL06中2-FL产量的优化

以酿酒酵母W303-1a基因组为模板，以引物SEC-F和SEC-R扩增出甘露糖磷酸异构酶(Phosphomannomutase)的序列，命名为Sec53，其氨基酸序列如SEQ ID No.19所示，其核苷酸序列如SEQ ID No.20所示。以引物PSA1-F和PSA1-R扩增出GDP-甘露糖焦磷酸酶(GDP-mannose pyrophosphorylase)的序列，命名为Psa1，其氨基酸序列如SEQ ID No.21所示，其核苷酸序列如SEQ ID No.22所示。

以pUMRI-A质粒为模板，10-s-F和10-p-R为引物扩增出双向启动子gal1,10。以酿酒酵母W303-1a总DNA为模板，以XbaI-ADH1-F和ADH1-sec-R引物扩增出adh1终止子序列，以CYC1-psa1-F和EcoRI-cyc1-R引物扩增出cyc1终止子序列。

通过SOE-PCR的方法，将以上片段依次连接，得到双向基因表达盒P_gal1-sec53-T_cyc1&P_gal10-psa1-T_adh1。并通过无缝连接的方法连在XbaI和EcoRI酶切后的载体pRS303上，得到重组载体pRS303-P_gal1-sec53-T_cyc1&P_gal10-psa1-T_adh1；所用引物序列如下：

重组载体pRS303-P_gal1-sec53-T_cyc1&P_gal10-psa1-T_adh1以PciI酶线性化，切胶回收。将线性化后的pRS303-P_gal1-sec53-T_cyc1&P_gal10-psa1-T_adh1通过醋酸锂转化法转入实施例3制备的重组菌株FL06中，整合位点为HIS3，涂布于色氨酸、尿嘧啶、亮氨酸、组氨酸缺陷的YNB固体平板上。转化子长出后挑取于色氨酸、尿嘧啶、亮氨酸、组氨酸缺陷的YNB液体培养基中，提取总DNA以引物XbaI-ADH1-F和EcoRI-cyc1-R进行PCR验证，正确的为阳性克隆子，命名为FL10。

将菌株于添加2％葡萄糖、2％半乳糖和0.4％乳糖的YP培养基中培养96h后，离心取上清为胞外产物，菌体冲洗两遍以后破碎，所得到的细胞裂解液为胞内产物。将胞外及胞内产物进行HPLC分析。并利用2-FL标准品进行定量分析，结果如图5所示，过表达甘露糖磷酸异构酶和GDP-甘露糖合酶，可以将2-FL的产量进一步提升至约3.5g/L。

以酿酒酵母W303-1a基因组为模板，以引物BamHI-PMI40-F和NheI-PMI40-R扩增出甘露糖-6-磷酸异构酶(Mannose-6-phosphate isomerase)的序列，命名为Pmi40，其氨基酸序列如SEQ ID No.23所示，其核苷酸序列如SEQ ID No.24所示。

通过无缝连接的方法将Pmi40连在BamHI和NheI酶切后的载体pUMRI-A上，得到重组载体pUMRI-A-Pgal10-pmi40-T_adh1。所用引物序列如下：

BamHI-PMI40-F	AAGGAGAAAAAACCCCGGATCCATGTCCAACAAGCTGTTCAG
		NheI-PMI40-R	GTTAGAGCGGATCTTAGCTAGCCTAATTTGGTTCCACAAAGG

重组载体pUMRI-A-Pgal10-pmi40-T_adh1以BsiWI酶线性化，切胶回收。将线性化后的pUMRI-A-Pgal10-pmi40-T_adh1通过醋酸锂转化法转入上述得到的重组菌株FL10中，整合位点为URA3，涂布于添加200μg/mLG418的YPD固体平板上。转化子长出后挑取于添加200μg/mLG418的YPD液体培养基中，提取总DNA以引物XbaI-ADH1-F和EcoRI-cyc1-R进行PCR验证，正确的为阳性克隆子，命名为FL11。

将菌株FL11于添加2％葡萄糖、2％半乳糖和0.4％乳糖的YP培养基中培养96h后，离心取上清为胞外产物，菌体冲洗两遍以后破碎，所得到的细胞裂解液为胞内产物。将胞外及胞内产物进行HPLC分析。并利用2-FL标准品进行定量分析，结果如图5所示，在过表达甘露糖磷酸异构酶和GDP-甘露糖合酶的基础上进一步表达果糖-甘露糖-6-磷酸异构酶，可以将2-FL的产量提升至约3.7g/L。

以酿酒酵母W303-1a基因组为模板，以引物EcoRI-POS5-F和SacI-POS5-R扩增出无线粒体信号肽的NADH激酶(NADHkinase)的序列，命名为sPos5，其氨基酸序列如SEQ IDNo.25所示，其核苷酸序列如SEQ ID No.26所示。

通过无缝连接的方法将sPos5编码序列连在EcoRI和SacI酶切后的载体pUMRI-A上，得到重组载体pUMRI-A-Pgal1-spos5-T_cyc1。测序正确后，利用BamHI和NheI酶切载体pUMRI-A-Pgal1-spos5-T_cyc1，通过无缝连接的方法将Pmi40的编码序列连接在pUMRI-A-Pgal1-spos5-T_cyc1上，得到重组载体pUMRI-A-P_gal1-spos5-T_cyc1&P_gal10-pmi40-T_adh1。所用引物序列如下：

EcoRI-POS5-F	GAATTTTTGAAAATTCGAATTCATGAGTACGTTGGATTCACATTC
		SacI-POS5-R	AGAATTGTTAATTAAGAGCTCTTAATCATTATCAGTCTGTCTCTTGGTC

重组载体pUMRI-A-P_gal1-spos5-T_cyc1&P_gal10-pmi40-T_adh1以BsiWI酶线性化，切胶回收。将线性化后的pUMRI-A-P_gal1-spos5-T_cyc1&P_gal10-pmi40-T_adh1通过醋酸锂转化法转入上述得到的重组菌株FL10中，整合位点为URA3，涂布于添加200μg/mLG418的YPD固体平板上。转化子长出后挑取于添加200μg/mLG418的YPD液体培养基中，提取总DNA以引物SacI-POS5-R和NheI-PMI40-R进行PCR验证，正确的为阳性克隆子，命名为FL18。

将菌株FL18于添加2％葡萄糖、2％半乳糖和0.4％乳糖的YP培养基中培养96h后，离心取上清为胞外产物，菌体冲洗两遍以后破碎，所得到的细胞裂解液为胞内产物。将胞外及胞内产物进行HPLC分析。结果如图6所示，高表达无线粒体信号肽的NADH激酶能够少量提高酿酒酵母中2-FL的产量，约为3.8g/L。比较FL18与FL11等数目细胞产生2-FL的能力，FL18可以产生约0.91g/L/OD的2-FL，而FL11仅能产生0.80g/L/OD的2-FL。上述实施例中所涉及菌株如下表所示：

上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

SEQUENCE LISTING

<110> 山东大学

<120> 一株优化的高产岩藻糖基乳糖的酿酒酵母菌株及其应用

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cagttcgtgg aaatggcggc agcacagctg ggcatcaaac tgcgctttga aggcacgggc 840

gttgaagaga agggcattgt ggtttccgtc accgggcatg acgcgccggg cgttaaaccg 900

ggtgatgtga ttatcgctgt tgacccgcgt tacttccgtc cggctgaagt tgaaacgctg 960

ctcggcgacc cgaccaaagc gcacgaaaaa ctgggctgga aaccggaaat caccctcaga 1020

gagatggtgt ctgaaatggt ggctaatgac ctcgaagcgg cgaaaaaaca ctctctgctg 1080

aaatctcacg gctacgacgt ggcgatcgcg ctggagtcat aa 1122

<210> 5

<211> 321

<212> PRT

<213> Escherichia coli

<400> 5

Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly

1 5 10 15

Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val

20 25 30

Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His

35 40 45

Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala

50 55 60

Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile

65 70 75 80

Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln

85 90 95

Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro

100 105 110

Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr

115 120 125

Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile

130 135 140

Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser

145 150 155 160

Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser

165 170 175

Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr

180 185 190

Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met

195 200 205

Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val

210 215 220

Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu

225 230 235 240

Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Glu Leu

245 250 255

Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe

260 265 270

Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr

275 280 285

Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly

290 295 300

Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg

305 310 315 320

Gly

<210> 6

<211> 966

<212> DNA

<213> Escherichia coli

<400> 6

atgagtaaac aacgagtttt tattgctggt catcgcggga tggtcggttc cgccatcagg 60

cggcagctcg aacagcgcgg tgatgtggaa ctggtattac gcacccgcga cgagctgaac 120

ctgctggaca gccgcgccgt gcatgatttc tttgccagcg aacgtattga ccaggtctat 180

ctggcggcgg cgaaagtggg cggcattgtt gccaacaaca cctatccggc ggatttcatc 240

taccagaaca tgatgattga gagcaacatc attcacgccg cgcatcagaa cgacgtgaac 300

aaactgctgt ttctcggatc gtcctgcatc tacccgaaac tggcaaaaca gccgatggca 360

gaaagcgagt tgttgcaggg cacgctggag ccgactaacg agccttatgc tattgccaaa 420

atcgccggga tcaaactgtg cgaatcatac aaccgccagt acggacgcga ttaccgctca 480

gtcatgccga ccaacctgta cgggccacac gacaacttcc acccgagtaa ttcgcatgtg 540

atcccagcat tgctgcgtcg cttccacgag gcgacggcac agaatgcgcc ggacgtggtg 600

gtatggggca gcggtacacc gatgcgcgaa tttctgcacg tcgatgatat ggcggcggcg 660

agcattcatg tcatggagct ggcgcatgaa gtctggctgg agaacaccca gccgatgttg 720

tcgcacatta acgtcggcac gggcgttgac tgcactatcc gcgagctggc gcaaaccatc 780

gccaaagtgg tgggttacaa aggccgggtg gtttttgatg ccagcaaacc ggatggcacg 840

ccgcgcaaac tgctggatgt gacgcgcctg catcagcttg gctggtatca cgaaatctca 900

ctggaagcgg ggcttgccag cacttaccag tggttccttg agaatcaaga ccgctttcgg 960

gggtaa 966

<210> 7

<211> 300

<212> PRT

<213> Helicobacter pylori

<400> 7

Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met

1 5 10 15

Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Ser Asn Thr Pro

20 25 30

Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asp Arg Lys Met Gln

35 40 45

Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Ala Lys Glu Ile

50 55 60

Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Asp Ala Leu

65 70 75 80

Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr

85 90 95

Glu Pro Lys Leu Leu Lys Pro Ser Arg Leu Thr Tyr Phe Phe Gly Tyr

100 105 110

Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln

115 120 125

Thr Phe Thr Leu Pro Pro Pro Pro Glu Asn Asn Lys Asn Asn Asn Lys

130 135 140

Lys Glu Glu Glu Tyr Gln Cys Lys Leu Ser Leu Ile Leu Ala Ala Lys

145 150 155 160

Asn Ser Val Phe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly

165 170 175

Cys Gln Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala

180 185 190

Lys Arg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Glu

195 200 205

Phe Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr

210 215 220

Arg Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser

225 230 235 240

Cys Gln His Gly Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala

245 250 255

Tyr Leu Ile Glu Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp

260 265 270

Leu Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu

275 280 285

Ser His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala

290 295 300

<210> 8

<211> 903

<212> DNA

<213> Helicobacter pylori

<400> 8

atggctttta aggtggtgca aatttgcgga gggcttggga atcaaatgtt tcaatacgct 60

ttcgctaaaa gtttgcaaaa acactctaat acgcctgtgc tgttagatat cacttctttt 120

gattggagcg ataggaaaat gcaattagaa cttttcccta ttgatttgcc ctatgcgagc 180

gcgaaagaaa tcgctatagc taaaatgcaa cacctcccca agctagtaag agacgcgctc 240

aaatgcatgg gatttgatag ggtgagtcaa gaaatcgttt ttgaatacga gcctaaattg 300

ctaaagccaa gccgcttgac ttattttttt ggctatttcc aagatccacg atactttgat 360

gctatatccc ctttaatcaa gcaaaccttc actctaccac caccaccaga aaataataag 420

aataataata aaaaagagga agaatatcag tgcaagcttt ctttgatttt agccgctaaa 480

aacagcgtgt ttgtgcatat aagaagaggg gattatgtgg ggattggctg tcagcttggt 540

attgactatc aaaaaaaggc gcttgagtat atggcaaagc gcgtgccaaa catggagctt 600

tttgtgtttt gcgaagactt agaattcacg caaaatcttg atcttggcta cccttttatg 660

gacatgacca ctagggataa agaagaagag gcgtattggg acatgctgct catgcaatct 720

tgtcagcatg gcattatcgc taatagcact tatagctggt gggcggccta tttgatagaa 780

aatccagaaa aaatcattat tggccccaaa cactggcttt ttgggcatga gaatatcctt 840

tgtaaggagt gggtgaaaat agaatcccat tttgaggtaa aatcccaaaa gtataacgct 900

taa 903

<210> 9

<211> 308

<212> PRT

<213> Acetobacter sp.

<400> 9

Met Ala Val Ser Pro Gln Glu Ser Lys Tyr Ser Ala His Val Ser Pro

1 5 10 15

Asp Lys Pro Leu Arg Ile Val Arg Leu Gly Gly Gly Leu Gly Asn Gln

20 25 30

Met Phe Gln Tyr Ala Phe Gly Leu Ala Ala Gly Asp Val Leu Trp Asp

35 40 45

Asn Thr Ser Phe Leu Thr Asn His Tyr Arg Ser Phe Asp Leu Gly Leu

50 55 60

Tyr Asn Ile Ser Gly Asp Phe Ala Ser Asn Glu Gln Ile Lys Lys Cys

65 70 75 80

Lys Asn Glu Ile Arg Phe Lys Asn Ile Leu Pro Arg Ser Ile Arg Lys

85 90 95

Lys Phe Asn Leu Gly Lys Phe Ile Tyr Leu Lys Thr Asn Arg Val Cys

100 105 110

Glu Arg Gln Ile Asn Arg Tyr Glu Pro Glu Leu Leu Ser Lys Asp Gly

115 120 125

Asp Val Tyr Tyr Asp Gly Val Phe Gln Thr Glu Lys Tyr Phe Lys Pro

130 135 140

Leu Arg Glu Arg Leu Leu His Asp Phe Thr Leu Thr Lys Pro Leu Asp

145 150 155 160

Ala Ala Asn Leu Asp Met Leu Ala Lys Ile Arg Ala Ala Asp Ala Val

165 170 175

Ala Val His Ile Arg Arg Gly Asp Tyr Leu Asn Pro Arg Ser Pro Phe

180 185 190

Thr Tyr Leu Asp Lys Asp Tyr Phe Leu Asn Ala Met Asp Tyr Ile Gly

195 200 205

Lys Arg Val Asp Lys Pro His Phe Phe Ile Phe Ser Ser Asp Thr Asp

210 215 220

Trp Val Arg Thr Asn Ile Gln Thr Ala Tyr Pro Gln Thr Ile Val Glu

225 230 235 240

Ile Asn Asp Glu Lys His Gly Tyr Phe Asp Leu Glu Leu Met Arg Asn

245 250 255

Cys Arg His Asn Ile Ile Ala Asn Ser Thr Phe Ser Trp Trp Gly Ala

260 265 270

Trp Leu Asn Thr Asn Pro Asp Lys Ile Val Val Ala Pro Lys Gln Trp

275 280 285

Phe Arg Pro Asp Ala Ala Glu Tyr Ser Gly Asp Ile Val Pro Asn Asp

290 295 300

Trp Ile Lys Leu

305

<210> 10

<211> 927

<212> DNA

<213> Acetobacter sp.

<400> 10

atggctgttt cacctcaaga aagcaaatat tccgcccatg tttccccgga taaaccgctg 60

cgtatcgtca ggctgggcgg agggctcggc aatcagatgt ttcaatatgc cttcggactg 120

gctgccggtg atgttttgtg ggacaatacc agctttttga ccaaccatta ccgcagtttt 180

gatttgggat tgtataacat cagcggagat tttgcctcaa atgaacaaat aaaaaaatgt 240

aaaaacgaaa tcagatttaa aaacattctt ccccgttcca tccgcaaaaa atttaatctg 300

ggaaaattta tttacctgaa aaccaaccgc gtctgcgaaa gacagattaa ccgttacgag 360

ccggaacttc taagcaaaga tggcgacgtt tattatgacg gagttttcca gaccgaaaaa 420

tattttaaac cgctgcgaga aaggctgttg catgatttta cgctgacaaa acctcttgat 480

gcagcgaatc ttgatatgct tgccaaaatc cgggctgccg atgccgttgc cgttcatatc 540

cgtcgcggcg actatcttaa tccgcgcagc ccttttactt atttggataa ggattatttt 600

ctgaatgcga tggattatat cgggaaacgc gttgataaac cgcatttctt tattttttcc 660

agtgatacag actgggttcg gactaatata caaacagcct atccgcagac tatcgttgaa 720

attaatgatg aaaaacacgg ctattttgac ttggaactga tgcggaactg ccgccataac 780

attatcgcca acagcacatt ttcgtggtgg ggagcctggc tgaatacaaa tccggacaag 840

attgtcgttg cgcctaagca atggttcagg ccggacgctg ccgaatattc cggtgatatc 900

gttcctaacg actggataaa gttataa 927

<210> 11

<211> 291

<212> PRT

<213> Bacillus cereus

<400> 11

Met Lys Ile Ile Gln Val Ser Ser Gly Leu Gly Asn Gln Met Phe Gln

1 5 10 15

Tyr Ala Leu Tyr Lys Lys Ile Ser Leu Asn Asp Asn Asp Val Phe Leu

20 25 30

Asp Ser Ser Thr Ser Tyr Met Met Tyr Lys Asn Gln His Asn Gly Tyr

35 40 45

Glu Leu Glu Arg Ile Phe His Ile Lys Pro Arg His Ala Gly Lys Glu

50 55 60

Ile Ile Asp Asn Leu Ser Asp Leu Asp Ser Glu Leu Ile Ser Arg Ile

65 70 75 80

Arg Arg Lys Leu Phe Gly Ala Lys Lys Ser Met Tyr Val Glu Leu Lys

85 90 95

Glu Phe Glu Tyr Asp Pro Ile Ile Phe Glu Lys Lys Glu Thr Tyr Phe

100 105 110

Lys Gly Tyr Trp Gln Asn Tyr Asn Tyr Phe Lys Asp Ile Glu Gln Glu

115 120 125

Leu Arg Lys Asp Phe Val Phe Thr Glu Lys Leu Asp Lys Arg Asn Glu

130 135 140

Lys Leu Ala Asn Glu Ile Arg Asn Lys Asn Ser Val Ser Ile His Ile

145 150 155 160

Arg Arg Gly Asp Tyr Tyr Leu Asn Lys Val Tyr Glu Glu Lys Phe Gly

165 170 175

Asn Ile Ala Asn Leu Glu Tyr Tyr Leu Lys Ala Ile Asn Leu Val Lys

180 185 190

Lys Lys Ile Glu Asp Pro Lys Phe Tyr Ile Phe Ser Asp Asp Ile Asp

195 200 205

Trp Ala Gln Lys Asn Ile Asn Leu Thr Asn Asp Val Val Tyr Ile Ser

210 215 220

His Asn Gln Gly Asn Glu Ser Tyr Lys Asp Met Gln Leu Met Ser Leu

225 230 235 240

Cys Lys His Asn Ile Ile Ala Asn Ser Thr Phe Ser Trp Trp Gly Ala

245 250 255

Phe Leu Asn Asn Asn Asp Asp Lys Ile Val Val Ala Pro Lys Lys Trp

260 265 270

Ile Asn Ile Lys Gly Leu Glu Lys Val Glu Leu Phe Pro Glu Asn Trp

275 280 285

Ile Thr Tyr

290

<210> 12

<211> 876

<212> DNA

<213> Bacillus cereus

<400> 12

atgaagatta ttcaggtgtc ttctggcctg ggcaatcaga tgtttcagta cgcactgtac 60

aagaaaattt ctctgaatga taatgatgtt tttctggact catcaacttc ttatatgatg 120

tacaaaaatc agcataatgg ttatgaactg gaaagaattt ttcatatcaa gccacgtcac 180

gcaggcaagg agattatcga taacctgtca gacctggact cagagctgat ctctcgtatc 240

cgtcgtaagc tgttcggcgc taaaaaatct atgtacgtgg aactgaaaga atttgaatat 300

gatccaatta tttttgagaa aaaagaaact tatttcaaag gttactggca gaactacaat 360

tattttaagg atattgaaca ggaattacgt aaagattttg tttttactga aaaattagat 420

aaaagaaatg aaaaactggc taacgaaatt agaaataaaa attcagtttc aattcacatt 480

agaagaggtg attattatct gaataaagtt tatgaggaaa aatttggtaa tattgcaaat 540

ctggaatatt atctgaaagc tattaatctg gttaaaaaga aaatcgaaga tccaaaattt 600

tatatttttt cagatgatat tgattgggct cagaaaaata ttaatctgac taatgatgtg 660

gtgtatattt ctcataacca gggtaacgag tcatacaagg acatgcagct gatgtctctg 720

tgcaaacata atattattgc taactctact ttctcttggt ggggcgcttt cctgaacaat 780

aatgacgaca aaattgtggt tgctcctaaa aaatggatta atatcaaagg tctggaaaaa 840

gttgaactgt ttccagaaaa ttggattact tattga 876

<210> 13

<211> 281

<212> PRT

<213> Bacteroides eggerthii

<400> 13

Met Arg Leu Ile Lys Met Thr Gly Gly Leu Gly Asn Gln Met Phe Ile

1 5 10 15

Tyr Ala Phe Tyr Leu Arg Met Lys Lys Arg His Thr Asn Thr Arg Ile

20 25 30

Asp Leu Ser Asp Met Met His Tyr Asn Val His His Gly Tyr Glu Met

35 40 45

His Arg Val Phe Asn Leu Pro Lys Thr Glu Phe Cys Ile Asn Gln Pro

50 55 60

Leu Lys Lys Val Ile Glu Phe Leu Phe Phe Lys Lys Ile Tyr Glu Arg

65 70 75 80

Lys Gln Asp Pro Ser Ser Leu Leu Pro Phe Asp Lys Lys Tyr Leu Trp

85 90 95

Pro Leu Leu Tyr Phe Lys Gly Phe Tyr Gln Ser Glu Arg Phe Phe Ala

100 105 110

Asp Met Glu Asn Asp Ile Arg Ile Ala Phe Thr Phe Asn Ser Asp Leu

115 120 125

Phe Asn Glu Lys Thr Gln Ala Met Leu Thr Gln Ile Lys His Asn Glu

130 135 140

His Ala Val Ser Leu His Ile Arg Arg Gly Asp Tyr Leu Glu Pro Lys

145 150 155 160

His Trp Lys Thr Thr Gly Ser Val Cys Gln Leu Pro Tyr Tyr Leu Asn

165 170 175

Ala Ile Thr Glu Met Asn Lys Arg Ile Glu Gln Pro Ser Tyr Tyr Val

180 185 190

Phe Ser Asp Asp Ile Ala Trp Val Lys Glu Asn Leu Pro Leu Pro Gln

195 200 205

Ala Val Phe Ile Asp Trp Asn Lys Gly Ala Glu Ser Trp Gln Asp Met

210 215 220

Met Leu Met Ser His Cys Arg His His Ile Ile Cys Asn Ser Thr Phe

225 230 235 240

Ser Trp Trp Gly Ala Trp Leu Asn Pro Arg Glu Asn Lys Thr Val Ile

245 250 255

Met Pro Glu Arg Trp Phe Gln His Cys Asp Thr Pro Asn Ile Tyr Pro

260 265 270

Asp Gly Trp Ile Lys Val Pro Val Asn

275 280

<210> 14

<211> 846

<212> DNA

<213> Bacteroides eggerthii

<400> 14

atgagactga ttaaaatgac aggcggcctg ggcaaccaaa tgtttatcta cgctttctat 60

ctacgaatga aaaaacgtca tacaaatact cggatcgatt tgtccgacat gatgcattac 120

aacgtccacc atggttatga aatgcaccgc gtgttcaatc ttcccaaaac agaattctgt 180

atcaaccaac ctttgaaaaa ggttatagaa tttcttttct tcaagaaaat ctatgaacgc 240

aaacaagacc catcaagtct cctcccattc gataaaaaat acttatggcc tttgctatac 300

tttaaaggat tctatcagtc ggaaaggttc tttgccgata tggaaaatga catacgtata 360

gcctttacat tcaactcaga cctgttcaat gagaagaccc aagcaatgct aacgcaaata 420

aagcataacg agcatgccgt ttcactacat atccgccggg gagattattt agaacccaaa 480

cattggaaaa caacaggcag tgtgtgccaa ctgccctatt acctcaatgc tattaccgaa 540

atgaacaaac ggatagagca gccttcctat tatgtttttt cggatgacat agcctgggta 600

aaggaaaacc taccgttgcc acaggccgtc ttcattgatt ggaataaagg tgcagaaagc 660

tggcaggaca tgatgctgat gagccattgc cgccaccaca tcatttgcaa cagtacgttt 720

agctggtggg gggcatggtt gaaccccaga gagaataaga cggtaattat gcccgaacgt 780

tggtttcaac attgtgatac gcccaacatc tatcctgacg gttggataaa agtacccgtt 840

aactga 846

<210> 15

<211> 290

<212> PRT

<213> Bacteroides uniformis

<400> 15

Met Lys Ile Val Leu Pro Cys Trp Gly Leu Gly Asn Val Met Phe Gln

1 5 10 15

Tyr Ala Phe Leu Cys Glu Leu Arg Phe Arg Gly Arg Glu Ala Cys Cys

20 25 30

Phe Leu Leu His Lys Lys Leu Arg Phe Glu His Asn Gly Tyr Glu Leu

35 40 45

Asp Lys Leu Phe Lys Val His Pro Tyr Lys Gly Leu Asn Ile Phe Gln

50 55 60

Arg Ile Tyr Ile Arg Leu Val Glu Phe Leu Gly Ala Met Glu Leu Pro

65 70 75 80

Tyr Phe Lys Leu Ile Ser Leu Phe Phe Lys Glu Val Ser Val Lys Glu

85 90 95

Asn Phe Ile Tyr Tyr Asp Glu Val Phe Gln His Pro His Glu Asn Cys

100 105 110

Tyr Phe Lys Gly Thr Trp Gln Ser Pro Leu Tyr Phe Val His Ala Lys

115 120 125

Gln Glu Ile Leu Asp Thr Phe Arg Phe Asp Lys Arg Met Ile Ser Ser

130 135 140

Tyr Thr Arg Asp Val Leu Asn Gln Ile Glu Lys Thr Pro Asn Ser Val

145 150 155 160

Ser Leu His Val Arg Arg Gly Asp Tyr Leu Lys Pro Asp Phe Glu Gly

165 170 175

Leu Ser Lys Cys Cys Pro Asn Asp Tyr Phe Glu Arg Ala Ile Gln Tyr

180 185 190

Met Lys Glu His Met Asp Thr Pro Val Phe Tyr Val Phe Ser Asp Asp

195 200 205

Met Asp Tyr Val Lys Glu Asn Ile Lys Leu Glu Asp Ala Phe Tyr Ile

210 215 220

Asp Gly Asn Arg Gly Asn Asp Ser Trp Gln Asp Met Phe Leu Met Ser

225 230 235 240

Ser Cys Asn His Asn Ile Ile Ala Asn Ser Thr Phe Ser Trp Trp Ser

245 250 255

Ala Phe Leu Asn Ser His Asp Asn Lys Ile Val Ile Ala Pro Lys Arg

260 265 270

Trp Trp Tyr Tyr Phe Glu Thr Asp Asp Val Val Pro Glu Glu Trp Ile

275 280 285

Arg Met

290

<210> 16

<211> 873

<212> DNA

<213> Bacteroides uniformis

<400> 16

atgaaaatag tattgccatg ttggggatta ggaaatgtta tgtttcagta cgcattttta 60

tgtgaactgc gttttagagg aagagaagct tgttgtttct tattacataa aaaattacgt 120

tttgaacata atggatatga gcttgataaa ttgtttaaag tacatcctta taaaggactt 180

aatattttcc aaaggatata tatacgttta gtagagttct tgggggctat ggaattacct 240

tattttaaat taatcagcct tttctttaaa gaggtaagtg ttaaagaaaa ttttatatat 300

tatgatgagg tatttcaaca tccacatgaa aattgctatt ttaaaggaac ttggcagagt 360

cctttgtatt ttgttcatgc caagcaagag atattggata catttcgatt tgacaaaaga 420

atgatttctt cttatacaag agatgtttta aaccaaatag aaaagactcc taacagtgta 480

agtttgcatg tacgtcgcgg tgattatttg aaaccagatt ttgaaggatt gagtaaatgt 540

tgtcctaacg attattttga aagggcaatt caatatatga aagagcatat ggataccccg 600

gttttctatg tcttttctga tgatatggat tatgtgaagg aaaatataaa gttggaagac 660

gctttctata ttgatggtaa tagggggaat gattcttggc aagatatgtt tctaatgagt 720

tcatgcaatc ataatattat agctaattct acatttagtt ggtggagtgc ttttcttaat 780

tcccatgaca ataaaattgt tattgcacca aaacgctggt ggtattattt tgagacggat 840

gatgttgtac cggaagaatg gataagaatg tga 873

<210> 17

<211> 313

<212> PRT

<213> Neocallimastix californiae

<400> 17

Met Lys Ile Leu Ile Phe Ser Val Ser Phe Ser Phe Phe Tyr Leu Leu

1 5 10 15

His Leu Phe Phe Ile Leu Tyr Tyr Ile Ile Ser Lys Ala Ser Lys Glu

20 25 30

Ile Arg Ile Val Lys Leu Cys Gly Gly Leu Gly Asn Gln Met Phe Gln

35 40 45

Tyr Ala Tyr Gly Lys Ser Leu Glu His Lys Leu Gln Glu Lys Val Leu

50 55 60

Phe Asp Val Ser Trp Tyr Lys Tyr Leu Asn Lys Lys Lys Asn Glu Lys

65 70 75 80

Leu Thr Lys Arg Glu Tyr Gly Leu Gly Ile Phe Asn Leu Lys Ile Ser

85 90 95

Phe Pro Thr Lys Lys Gln Leu Lys Lys Cys Asn Asn Lys Thr Phe Glu

100 105 110

Lys Lys Ser Tyr Ile Tyr Asp Glu Glu Leu Leu Gln Asn Lys Gly Ser

115 120 125

Ser Tyr Tyr Val Gly Tyr Phe Gln Asn Glu Lys Tyr Phe Lys Asp Ile

130 135 140

Lys Asp Asn Ile Lys Lys Ile Tyr Thr Phe Pro Lys Ile His Asp Thr

145 150 155 160

Asp Lys Phe Asn Gln Gln Trp Ile Asn Lys Ile Lys Asn Val Lys Asn

165 170 175

Ser Val Phe Ile His Ile Arg Arg Ala Asp Tyr Ile Tyr Leu Asp Gly

180 185 190

Trp Val Leu Ser Met Asp Tyr Tyr Lys Lys Ala Ile Glu Tyr Ile Lys

195 200 205

Lys Asn Val Glu Asn Pro Thr Phe Phe Ile Phe Cys Tyr Gln Cys Lys

210 215 220

Asp Tyr Val Glu Glu Gln Phe Lys Leu Asp Asp Thr Ile Gln Phe Ile

225 230 235 240

Gly Glu Thr Asn Ser Ile Asn Asn Glu Asn Trp Lys Asp Met Val Leu

245 250 255

Met Lys Glu Cys Lys Tyr Ala Ile Ile Ala Asn Ser Ser Phe Ser Trp

260 265 270

Trp Ala Ala Trp Leu Gly Arg Ala Asn Glu Glu Gly Ile Val Ile Ala

275 280 285

Pro Ser Pro Phe Ile Lys Asn Asn Asp Glu Ile Ile Cys Asp Asn Trp

290 295 300

Ile Lys Ile Asn Ser Asn Asn Ser Ser

305 310

<210> 18

<211> 942

<212> DNA

<213> Neocallimastix californiae

<400> 18

atgaaaatat taatattctc tgtttctttt tcctttttct atttattaca tttatttttc 60

atactttact atattatttc taaagcatcc aaagaaataa gaatagtaaa actatgtgga 120

ggtttaggaa atcaaatgtt tcaatatgct tatgggaaat ctcttgaaca caaattacaa 180

gaaaaggtat tatttgatgt ttcatggtat aaatatttga ataaaaagaa aaatgaaaaa 240

ttaactaaaa gggaatatgg attgggtata tttaatttaa aaatatcttt tcctactaaa 300

aaacaactaa aaaaatgtaa taataaaaca tttgaaaaga agagttacat atatgatgaa 360

gaattattac aaaataaagg atcatcgtat tatgtgggat attttcaaaa tgaaaaatat 420

tttaaagata ttaaagataa tataaaaaaa atatatacat ttccaaaaat tcatgatact 480

gataaattta atcaacaatg gataaataaa ataaaaaacg tcaaaaattc cgtctttatt 540

catatcagaa gagcagatta tatatattta gatggatggg ttctttcaat ggattattat 600

aaaaaggcaa ttgagtatat aaaaaaaaat gtagaaaatc ctacattttt catattttgt 660

taccaatgta aagattatgt tgaagagcaa ttcaaattag atgatacaat tcagtttata 720

ggagaaacaa attcaattaa taatgaaaat tggaaagata tggttttaat gaaggaatgt 780

aaatatgcaa taatagcaaa ttcatcattt agttggtggg cagcatggct tggaagagcc 840

aatgaagaag gcattgtaat tgctccttct ccatttatta aaaataatga tgaaataata 900

tgtgataatt ggattaaaat taatagtaat aattcttcat ag 942

<210> 19

<211> 254

<212> PRT

<213> Saccharomyces cerevisiae

<400> 19

Met Ser Ile Ala Glu Phe Ala Tyr Lys Glu Lys Pro Glu Thr Leu Val

1 5 10 15

Leu Phe Asp Val Asp Gly Thr Leu Thr Pro Ala Arg Leu Thr Val Ser

20 25 30

Glu Glu Val Arg Lys Thr Leu Ala Lys Leu Arg Asn Lys Cys Cys Ile

35 40 45

Gly Phe Val Gly Gly Ser Asp Leu Ser Lys Gln Leu Glu Gln Leu Gly

50 55 60

Pro Asn Val Leu Asp Glu Phe Asp Tyr Ser Phe Ser Glu Asn Gly Leu

65 70 75 80

Thr Ala Tyr Arg Leu Gly Lys Glu Leu Ala Ser Gln Ser Phe Ile Asn

85 90 95

Trp Leu Gly Glu Glu Lys Tyr Asn Lys Leu Ala Val Phe Ile Leu Arg

100 105 110

Tyr Leu Ser Glu Ile Asp Leu Pro Lys Arg Arg Gly Thr Phe Leu Glu

115 120 125

Phe Arg Asn Gly Met Ile Asn Val Ser Pro Ile Gly Arg Asn Ala Ser

130 135 140

Thr Glu Glu Arg Asn Glu Phe Glu Arg Tyr Asp Lys Glu His Gln Ile

145 150 155 160

Arg Ala Lys Phe Val Glu Ala Leu Lys Lys Glu Phe Pro Asp Tyr Gly

165 170 175

Leu Thr Phe Ser Ile Gly Gly Gln Ile Ser Phe Asp Val Phe Pro Ala

180 185 190

Gly Trp Asp Lys Thr Tyr Cys Leu Gln His Val Glu Lys Asp Gly Phe

195 200 205

Lys Glu Ile His Phe Phe Gly Asp Lys Thr Met Val Gly Gly Asn Asp

210 215 220

Tyr Glu Ile Phe Val Asp Glu Arg Thr Ile Gly His Ser Val Gln Ser

225 230 235 240

Pro Asp Asp Thr Val Lys Ile Leu Thr Glu Leu Phe Asn Leu

245 250

<210> 20

<211> 765

<212> DNA

<213> Saccharomyces cerevisiae

<400> 20

atgagtatcg ctgaattcgc ttacaaggaa aaaccagaaa ctttggtttt attcgatgtt 60

gatggtacct tgacaccagc cagattaact gtttctgaag aagttagaaa aactttggcc 120

aagttgagaa acaagtgctg cattggtttt gtcggtggtt ctgacttaag caagcaatta 180

gaacagttag gcccaaacgt tttagatgaa tttgactatt ctttctctga aaatggtttg 240

accgcctaca gattaggtaa ggaattagct tctcaatcct tcatcaactg gctcggtgag 300

gaaaaataca ataaattggc cgtcttcatt ttgagatatc tatctgaaat tgacttgcca 360

aagagaagag gtactttctt ggaatttaga aatggtatga tcaacgtttc cccaattggt 420

agaaatgctt ctactgagga aagaaacgaa ttcgaaagat acgataagga acaccaaatc 480

agagccaagt tcgttgaagc tttgaaaaag gaattcccag actacggttt gactttctcc 540

attggtggcc aaatctcttt cgacgttttc cccgctggtt gggataagac ctactgtttg 600

caacacgttg aaaaagatgg tttcaaggaa attcatttct ttggtgacaa gactatggtc 660

ggtggtaacg attacgaaat ttttgtcgat gaaagaacca tcggacattc agtacaatcc 720

cctgatgaca ccgtcaaaat tttgactgaa ctattcaact tatag 765

<210> 21

<211> 361

<212> PRT

<213> Saccharomyces cerevisiae

<400> 21

Met Lys Gly Leu Ile Leu Val Gly Gly Tyr Gly Thr Arg Leu Arg Pro

1 5 10 15

Leu Thr Leu Thr Val Pro Lys Pro Leu Val Glu Phe Gly Asn Arg Pro

20 25 30

Met Ile Leu His Gln Ile Glu Ala Leu Ala Asn Ala Gly Val Thr Asp

35 40 45

Ile Val Leu Ala Val Asn Tyr Arg Pro Glu Val Met Val Glu Thr Leu

50 55 60

Lys Lys Tyr Glu Lys Glu Tyr Gly Val Asn Ile Thr Phe Ser Val Glu

65 70 75 80

Thr Glu Pro Leu Gly Thr Ala Gly Pro Leu Lys Leu Ala Glu Asp Val

85 90 95

Leu Lys Lys Asp Asn Ser Pro Phe Phe Val Leu Asn Ser Asp Val Ile

100 105 110

Cys Glu Tyr Pro Phe Lys Glu Leu Ala Asp Phe His Lys Ala His Gly

115 120 125

Gly Lys Gly Thr Ile Val Ala Thr Lys Val Asp Glu Pro Ser Lys Tyr

130 135 140

Gly Val Ile Val His Asp Ile Ala Thr Pro Asn Leu Ile Asp Arg Phe

145 150 155 160

Val Glu Lys Pro Lys Glu Phe Val Gly Asn Arg Ile Asn Ala Gly Leu

165 170 175

Tyr Ile Leu Asn Pro Glu Val Ile Asp Leu Ile Glu Met Lys Pro Thr

180 185 190

Ser Ile Glu Lys Glu Thr Phe Pro Ile Leu Val Glu Glu Lys Gln Leu

195 200 205

Tyr Ser Phe Asp Leu Glu Gly Phe Trp Met Asp Val Gly Gln Pro Lys

210 215 220

Asp Phe Leu Ser Gly Thr Val Leu Tyr Leu Asn Ser Leu Ala Lys Arg

225 230 235 240

Gln Pro Lys Lys Leu Ala Thr Gly Ala Asn Ile Val Gly Asn Ala Leu

245 250 255

Ile Asp Pro Thr Ala Lys Ile Ser Ser Thr Ala Lys Ile Gly Pro Asp

260 265 270

Val Val Ile Gly Pro Asn Val Thr Ile Gly Asp Gly Val Arg Ile Thr

275 280 285

Arg Ser Val Val Leu Cys Asn Ser Thr Ile Lys Asn His Ser Leu Val

290 295 300

Lys Ser Thr Ile Val Gly Trp Asn Ser Thr Val Gly Gln Trp Cys Arg

305 310 315 320

Leu Glu Gly Val Thr Val Leu Gly Asp Asp Val Glu Val Lys Asp Glu

325 330 335

Ile Tyr Ile Asn Gly Gly Lys Val Leu Pro His Lys Ser Ile Ser Asp

340 345 350

Asn Val Pro Lys Glu Ala Ile Ile Met

355 360

<210> 22

<211> 1086

<212> DNA

<213> Saccharomyces cerevisiae

<400> 22

atgaaaggtt taattttagt cggtggttac ggtaccagat tgagaccttt aactttgacc 60

gttccaaagc cactggttga attcggtaat agaccaatga ttttacacca aatcgaggct 120

ttagccaacg ctggtgttac tgacatcgtt cttgctgtta attacagacc agaagtcatg 180

gtggaaactt tgaagaagta cgaaaaggaa tatggtgtta acatcacttt ctctgtagaa 240

actgaaccat taggtactgc aggtccattg aaattggctg aagatgtttt gaagaaggac 300

aactctccat ttttcgtcct aaactccgac gtcatttgcg aatatccatt caaggaattg 360

gctgacttcc acaaagctca cggtggtaaa ggtaccattg ttgctaccaa ggtcgacgaa 420

ccttctaaat acggtgtcat tgtccatgat atagctactc caaacttgat tgacagattt 480

gttgaaaagc caaaggaatt tgttggtaac agaattaacg ccggtttgta cattttaaac 540

ccagaagtca ttgacttgat tgaaatgaag ccaacttcaa ttgaaaagga aactttccca 600

atcttggtcg aagaaaaaca actatattcc ttcgatttgg aaggtttctg gatggatgtt 660

ggtcaaccaa aggacttctt gtctggtacc gttctttact tgaactcttt ggccaagaga 720

caaccaaaaa aattggctac aggtgccaac attgttggta atgccttgat cgacccaacc 780

gctaagattt cctccactgc taagattggc ccagacgtgg ttatcggtcc taatgtcacc 840

atcggtgatg gtgttagaat caccagatct gttgttttgt gcaactccac catcaagaac 900

cactccttgg tcaaatctac catcgtaggc tggaactcta ccgttggtca atggtgtcgt 960

ttggaaggtg tcactgtctt gggtgacgac gttgaagtta aggacgaaat ctacatcaac 1020

ggtggtaaag tcttacctca taagtctatc tccgataatg ttccaaagga agctattatt 1080

atgtga 1086

<210> 23

<211> 429

<212> PRT

<213> Saccharomyces cerevisiae

<400> 23

Met Ser Asn Lys Leu Phe Arg Leu Asp Ala Gly Tyr Gln Gln Tyr Asp

1 5 10 15

Trp Gly Lys Ile Gly Ser Ser Ser Ala Val Ala Gln Phe Ala Ala His

20 25 30

Ser Asp Pro Ser Val Gln Ile Glu Gln Asp Lys Pro Tyr Ala Glu Leu

35 40 45

Trp Met Gly Thr His Ser Lys Met Pro Ser Tyr Asn His Glu Ser Lys

50 55 60

Glu Ser Leu Arg Asp Ile Ile Ser Lys Asn Pro Ser Ala Met Leu Gly

65 70 75 80

Lys Asp Ile Ile Asp Lys Phe His Ala Thr Asn Glu Leu Pro Phe Leu

85 90 95

Phe Lys Val Leu Ser Ile Glu Lys Val Leu Ser Ile Gln Ala His Pro

100 105 110

Asp Lys Ala Leu Gly Lys Ile Leu His Ala Gln Asp Pro Lys Asn Tyr

115 120 125

Pro Asp Asp Asn His Lys Pro Glu Met Ala Ile Ala Val Thr Asp Phe

130 135 140

Glu Gly Phe Cys Gly Phe Lys Pro Leu Gln Glu Ile Ala Asp Glu Leu

145 150 155 160

Lys Arg Ile Pro Glu Leu Arg Asn Ile Val Gly Glu Glu Thr Ser Arg

165 170 175

Asn Phe Ile Glu Asn Ile Gln Pro Ser Ala Gln Lys Gly Ser Pro Glu

180 185 190

Asp Glu Gln Asn Lys Lys Leu Leu Gln Ala Val Phe Ser Arg Val Met

195 200 205

Asn Ala Ser Asp Asp Lys Ile Lys Ile Gln Ala Arg Ser Leu Val Glu

210 215 220

Arg Ser Lys Asn Ser Pro Ser Asp Phe Asn Lys Pro Asp Leu Pro Glu

225 230 235 240

Leu Ile Gln Arg Leu Asn Lys Gln Phe Pro Asp Asp Val Gly Leu Phe

245 250 255

Cys Gly Cys Leu Leu Leu Asn His Cys Arg Leu Asn Ala Gly Glu Ala

260 265 270

Ile Phe Leu Arg Ala Lys Asp Pro His Ala Tyr Ile Ser Gly Asp Ile

275 280 285

Met Glu Cys Met Ala Ala Ser Asp Asn Val Val Arg Ala Gly Phe Thr

290 295 300

Pro Lys Phe Lys Asp Val Lys Asn Leu Val Ser Met Leu Thr Tyr Thr

305 310 315 320

Tyr Asp Pro Val Glu Lys Gln Lys Met Gln Pro Leu Lys Phe Asp Arg

325 330 335

Ser Ser Gly Asn Gly Lys Ser Val Leu Tyr Asn Pro Pro Ile Glu Glu

340 345 350

Phe Ala Val Leu Glu Thr Thr Phe Asp Glu Lys Leu Gly Gln Arg His

355 360 365

Phe Glu Gly Val Asp Gly Pro Ser Ile Leu Ile Thr Thr Lys Gly Asn

370 375 380

Gly Tyr Ile Lys Ala Asp Gly Gln Lys Leu Lys Ala Glu Pro Gly Phe

385 390 395 400

Val Phe Phe Ile Ala Pro His Leu Pro Val Asp Leu Glu Ala Glu Asp

405 410 415

Glu Ala Phe Thr Thr Tyr Arg Ala Phe Val Glu Pro Asn

420 425

<210> 24

<211> 1290

<212> DNA

<213> Saccharomyces cerevisiae

<400> 24

atgtccaaca agctgttcag gttagatgca ggttaccaac aatacgactg gggtaaaatc 60

ggctcttctt cagctgtcgc tcaatttgct gcccattctg acccctctgt tcaaattgaa 120

caagataaac catatgcaga gttatggatg ggtacccaca gcaagatgcc ttcctacaac 180

catgagtcta aggaatccct gagagatatc atctccaaga acccctctgc catgttaggt 240

aaggacatta ttgataagtt ccacgccaca aatgaattgc ccttcctttt caaagttttg 300

tccattgaaa aagtcttgtc tattcaagca catcccgaca aagccttggg taaaatattg 360

cacgctcaag atcctaagaa ctatcctgat gataatcaca aacctgaaat ggccatcgct 420

gtgactgact ttgaaggttt ctgcgggttc aaacctttgc aagagattgc agatgaattg 480

aaacgtattc ctgaattacg caacattgtt ggtgaagaaa cttccaggaa ttttattgag 540

aacattcaac cttctgctca gaaaggttcc ccagaagatg agcaaaacaa aaagctattg 600

caagctgttt tcagcagggt catgaacgct tcggatgaca aaatcaagat tcaagctcgc 660

tccttggtcg aaagatcaaa gaattctcca tcagacttta acaaacctga tttaccagaa 720

ttaattcaaa gactgaataa acagttccct gatgacgtgg gtttgttttg tggatgttta 780

ttgttgaatc actgcagatt gaatgctggt gaagccatct ttttaagagc taaggatcct 840

cacgcctata taagcggtga tattatggaa tgtatggctg cttctgacaa cgtagttaga 900

gcaggcttca ctccaaaatt caaggatgtt aaaaacttgg tctccatgtt aacctataca 960

tatgatcctg tggaaaagca aaaaatgcag cctttaaagt tcgacaggtc ctctggtaac 1020

ggtaagtcag ttttatataa ccctccaatc gaagaatttg ctgtattgga gactactttt 1080

gatgagaaac ttggtcaaag gcattttgaa ggtgttgatg gtccaagtat cttaatcact 1140

acaaaaggta atggttacat taaagcagat ggccaaaaat tgaaagctga acccggtttt 1200

gtctttttca tcgctccaca tttgcctgtt gatttggaag ctgaagatga ggcgtttact 1260

acctatagag cctttgtgga accaaattag 1290

<210> 25

<211> 398

<212> PRT

<213> Saccharomyces cerevisiae

<400> 25

Met Ser Thr Leu Asp Ser His Ser Leu Lys Leu Gln Ser Gly Ser Lys

1 5 10 15

Phe Val Lys Ile Lys Pro Val Asn Asn Leu Arg Ser Ser Ser Ser Ala

20 25 30

Asp Phe Val Ser Pro Pro Asn Ser Lys Leu Gln Ser Leu Ile Trp Gln

35 40 45

Asn Pro Leu Gln Asn Val Tyr Ile Thr Lys Lys Pro Trp Thr Pro Ser

50 55 60

Thr Arg Glu Ala Met Val Glu Phe Ile Thr His Leu His Glu Ser Tyr

65 70 75 80

Pro Glu Val Asn Val Ile Val Gln Pro Asp Val Ala Glu Glu Ile Ser

85 90 95

Gln Asp Phe Lys Ser Pro Leu Glu Asn Asp Pro Asn Arg Pro His Ile

100 105 110

Leu Tyr Thr Gly Pro Glu Gln Asp Ile Val Asn Arg Thr Asp Leu Leu

115 120 125

Val Thr Leu Gly Gly Asp Gly Thr Ile Leu His Gly Val Ser Met Phe

130 135 140

Gly Asn Thr Gln Val Pro Pro Val Leu Ala Phe Ala Leu Gly Thr Leu

145 150 155 160

Gly Phe Leu Ser Pro Phe Asp Phe Lys Glu His Lys Lys Val Phe Gln

165 170 175

Glu Val Ile Ser Ser Arg Ala Lys Cys Leu His Arg Thr Arg Leu Glu

180 185 190

Cys His Leu Lys Lys Lys Asp Ser Asn Ser Ser Ile Val Thr His Ala

195 200 205

Met Asn Asp Ile Phe Leu His Arg Gly Asn Ser Pro His Leu Thr Asn

210 215 220

Leu Asp Ile Phe Ile Asp Gly Glu Phe Leu Thr Arg Thr Thr Ala Asp

225 230 235 240

Gly Val Ala Leu Ala Thr Pro Thr Gly Ser Thr Ala Tyr Ser Leu Ser

245 250 255

Ala Gly Gly Ser Ile Val Ser Pro Leu Val Pro Ala Ile Leu Met Thr

260 265 270

Pro Ile Cys Pro Arg Ser Leu Ser Phe Arg Pro Leu Ile Leu Pro His

275 280 285

Ser Ser His Ile Arg Ile Lys Ile Gly Ser Lys Leu Asn Gln Lys Pro

290 295 300

Val Asn Ser Val Val Lys Leu Ser Val Asp Gly Ile Pro Gln Gln Asp

305 310 315 320

Leu Asp Val Gly Asp Glu Ile Tyr Val Ile Asn Glu Val Gly Thr Ile

325 330 335

Tyr Ile Asp Gly Thr Gln Leu Pro Thr Thr Arg Lys Thr Glu Asn Asp

340 345 350

Phe Asn Asn Ser Lys Lys Pro Lys Arg Ser Gly Ile Tyr Cys Val Ala

355 360 365

Lys Thr Glu Asn Asp Trp Ile Arg Gly Ile Asn Glu Leu Leu Gly Phe

370 375 380

Asn Ser Ser Phe Arg Leu Thr Lys Arg Gln Thr Asp Asn Asp

385 390 395

<210> 26

<211> 1197

<212> DNA

<213> Saccharomyces cerevisiae

<400> 26

atgagtacgt tggattcaca ttccctaaag ttacagagcg gctcgaagtt tgtaaaaata 60

aagccagtaa ataacttgag gagtagttca tcagcagatt tcgtgtcccc accaaattcc 120

aaattacaat ctttaatctg gcagaaccct ttacaaaatg tttatataac taaaaaacca 180

tggactccat ccacaagaga agcgatggtt gaattcataa ctcatttaca tgagtcatac 240

cccgaggtga acgtcattgt tcaacccgat gtggcagaag aaatttccca ggatttcaaa 300

tctcctttgg agaatgatcc caaccgacct catatacttt atactggtcc tgaacaagat 360

atcgtaaaca gaacagactt attggtgaca ttgggaggtg atgggactat tttacacggc 420

gtatcaatgt tcggaaatac gcaagttcct ccggttttag catttgctct gggcactctg 480

ggctttctat caccgtttga ttttaaggag cataaaaagg tctttcagga agtaatcagc 540

tctagagcca aatgtttgca tagaacacgg ctagaatgtc atttgaaaaa aaaggatagc 600

aactcatcta ttgtgaccca tgctatgaat gacatattct tacatagggg taattcccct 660

catctcacta acctggacat tttcattgat ggggaatttt tgacaagaac gacagcagat 720

ggtgttgcat tggccactcc aacgggttcc acagcatatt cattatcagc aggtggatct 780

attgtttccc cattagtccc tgctatttta atgacaccaa tttgtcctcg ctctttgtca 840

ttccgaccac tgattttgcc tcattcatcc cacattagga taaagatagg ttccaaattg 900

aaccaaaaac cagtcaacag tgtggtaaaa ctttctgttg atggtattcc tcaacaggat 960

ttagatgttg gtgatgaaat ttatgttata aatgaggtcg gcactatata catagatggt 1020

actcagcttc cgacgacaag aaaaactgaa aatgacttta ataattcaaa aaagcctaaa 1080

aggtcaggga tttattgtgt cgccaagacc gagaatgact ggattagagg aatcaatgaa 1140

cttttaggat tcaattctag ctttaggctg accaagagac agactgataa tgattaa 1197

Claims

1.一株高产2’-岩藻糖基乳糖的酿酒酵母工程菌株，其特征在于，

所述酿酒酵母工程菌株中含有重组的乳糖透性酶(Lactosepermease)、重组的GDP-甘露糖脱水酶(GDP-mannose-4,6-dehydratase)、重组的GDP-岩藻糖合酶(GDP-L-fucosesynthase)以及重组的α-1,2-岩藻糖基转移酶(α-1,2fucosyltransferase)，所述α-1,2-岩藻糖基转移酶来源于蜡样芽孢杆菌(Bacillus cereus)；

所述酿酒酵母工程菌株还包含重组的甘露糖磷酸异构酶(Phosphomannomutase)和重组的GDP-甘露糖焦磷酸酶(GDP-mannose pyrophosphorylase)。

2.根据权利要求1所述的酿酒酵母工程菌株，其特征在于，所述酿酒酵母工程菌株还包含重组的甘露糖-6-磷酸异构酶(Mannose-6-phosphate isomerase)。

3.根据权利要求2所述的酿酒酵母工程菌株，其特征在于，所述酿酒酵母工程菌株还包含重组的无线粒体信号肽的NADH激酶(NADHkinase)。

4.根据权利要求1-3任一所述的酿酒酵母工程菌株，其特征在于，所述α-1,2-岩藻糖基转移酶的氨基酸序列如SEQ ID No.11所示；

优选的，所述乳糖透性酶来源于乳酸克鲁维斯酵母；更优选的，所述乳糖透性酶的氨基酸序列如SEQ ID No.1所示；

优选的，所述GDP-甘露糖脱水酶和GDP-岩藻糖合酶来源于大肠杆菌；更优选的，所述GDP-甘露糖脱水酶的氨基酸序列如SEQ ID No.3所示，所述GDP-岩藻糖合酶的氨基酸序列如SEQ ID No.5所示。

5.根据权利要求1-3任一所述的酿酒酵母工程菌株，其特征在于，所述甘露糖磷酸异构酶和GDP-甘露糖焦磷酸酶来源于酿酒酵母；优选的，所述甘露糖磷酸异构酶的氨基酸序列如SEQ ID No.19所示，所述GDP-甘露糖焦磷酸酶的氨基酸序列如SEQ ID No.21所示。

6.根据权利要求2-3任一所述的酿酒酵母工程菌株，其特征在于，所述甘露糖-6-磷酸异构酶来源于酿酒酵母；优选的，所述甘露糖-6-磷酸异构酶的氨基酸序列如SEQ ID No.23所示。

7.根据权利要求3所述的酿酒酵母工程菌株，其特征在于，所述NADH激酶来源于酿酒酵母；优选的，所述NADH激酶的氨基酸序列如SEQ ID No.25所示。

8.根据权利要求1-7任一所述的酿酒酵母工程菌株，其特征在于，所述酿酒酵母工程菌株的出发菌株为酿酒酵母W303-1a。

9.权利要求1-8任一所述的酿酒酵母工程菌株的制备方法，其特征在于，所述方法包括将所述目的基因重组到酿酒酵母中的步骤；

优选的，所述方法包括将所述目的基因整合到酿酒酵母的基因组中的步骤；更优选的，所述目的基因的整合位点包括TRP1、URA3、LEU2或HIS3。

10.权利要求1-8任一所述的酿酒酵母工程菌株在生产2’-岩藻糖基乳糖中的应用。