CN113249240A

CN113249240A - 一种高产羟基酪醇的酿酒酵母及其构建方法

Info

Publication number: CN113249240A
Application number: CN202110546281.0A
Authority: CN
Inventors: 罗云孜; 刘化一
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-08-13
Anticipated expiration: 2041-05-19
Also published as: CN113249240B

Abstract

本发明涉及微生物基因工程技术领域，公开了一种高产羟基酪醇的酿酒酵母及其构建方法。本发明所述高产羟基酪醇的酿酒酵母在能够合成酪醇的酿酒酵母基础上表达特定来源的HpaB和HpaC，由此实现羟基酪醇的制备和高产。本发明主要选择来源于铜绿假单胞菌的4‑羟基苯乙酸‑3‑羟化酶，并结合其他特定来源的核黄素氧化还原酶，将其转入到能够生产酪醇的酿酒酵母底盘细胞中，实现了羟基酪醇产量的提高；在此基础上进一步进行多种改造，使得所述酿酒酵母的羟基酪醇的产量高达1120mg/L，为微生物高效发酵生产羟基酪醇提供了新的途径。

Description

一种高产羟基酪醇的酿酒酵母及其构建方法

技术领域

本发明涉及微生物基因工程技术领域，具体的说是涉及一种高产羟基酪醇的酿酒酵母及其构建方法。

背景技术

羟基酪醇(3,4-二羟基苯乙醇)在食品、保健品、化妆品方面有广泛的应用，具有广阔的市场价值。目前从植物中提取羟基酪醇的产量低，纯化复杂。化学合成羟基酪醇需要昂贵的催化剂。采用微生物发酵的方式生产羟基酪醇具有对自然依赖低、不需要昂贵的催化剂且纯化相对容易等优势，因此一株可以高产羟基酪醇的工程菌具有极大的开发潜力。有现有技术采用来源于大肠杆菌(Escherichia coil)的4-羟基苯乙酸-3-羟化酶HpaB/核黄素氧化还原酶HpaC将酪醇转化为羟基酪醇；但是根据文献《Overproduction ofhydroxytyrosol in Saccharomyces cerevisiae by heterologous overexpression ofthe Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase》的记载，酿酒酵母中表达Ec来源的HpaB/HpaC，1mM酪醇仅能转化为4.6mg/L羟基酪醇，效率低下。目前尚未寻找到能够在酿酒酵母中将酪醇高效转化为羟基酪醇的酿酒酵母工程菌。

发明内容

有鉴于此，本发明的目的在于提供一种高产羟基酪醇的酿酒酵母及其构建方法，使得所述酿酒酵母可以显著提高羟基酪醇的产量。

为了实现上述目的，本发明提供如下技术方案：

一种高产羟基酪醇的酿酒酵母，在能够合成酪醇的酿酒酵母基础上进一步表达如下任意组合的HpaB和HpaC：

(1)来源于铜绿假单胞菌(Pseudomonas aeruginosa)的HpaB(PaHpaB)，以及来源于肠沙门氏菌(Salmonella enterica)、铜绿假单胞菌或大肠杆菌(Escherichia coli)的HpaC(SeHpaC、PaHpaC和EcHpaC)；或

(2)来源于大肠杆菌的HpaB(EcHpaB)和来源于铜绿假单胞菌的HpaC。

本发明在能够合成酪醇的酿酒酵母中表达上述来源组合的的HpaB和HpaC，能够高效地将酿酒酵母中酪醇转化为羟基酪醇，并相对于其他组合在羟基酪醇产量上有明显的提升。

在本发明具体实施方式中，所述PaHpaB既可以是野生型，也可以是具有Q212D突变的突变型；所述PaHpaB的野生型氨基酸序列如SEQ ID No.1所示，其对应的核苷酸序列包括但不限于SEQ ID No.2所示；所述PaHpaB的Q212D突变型氨基酸序列在SEQ ID No.1所示序列基础上第212位氨基酸变更为D，其对应的核苷酸序列在SEQ ID No.2所示序列基础上第634-636位碱基变更为GAC，但并不仅限于此序列；其他来源的野生型HpaB和HpaC序列如下：

SeHpaC、PaHpaC和EcHpaC氨基酸序列依次如SEQ ID No.3-5所示，对应的核苷酸序列依次如SEQ ID No.6-8所示；EcHpaB氨基酸序列如SEQ ID No.9所示，对应的核苷酸序列如SEQ ID No.10所示。

本发明所述能够合成酪醇的酿酒酵母可参照现有技术进行构建，本发明给出了一种较佳的构建方式：

(1)过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L和ARO7^G141S突变体，以及敲除酿酒酵母PHA2和PDC1的表达；

(2)过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L、ARO7^G141S和ARO3^D154N突变体，以及敲除酿酒酵母PHA2和PDC1的表达；或

(3)过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L、ARO7^G141S和ARO3^D154N突变体，过表达来源于大肠杆菌的TyrA^{M53I V354V}双突变体，以及敲除酿酒酵母PHA2和PDC1的表达。

过表达可以通过过表达载体的形式来实现，或者提高目标基因的表达量，敲除酿酒酵母PHA2和PDC1的表达可以通过CRIPSR-Cas9的方法敲除。

在本发明具体实施方式中，采用本领域常用的CEN.PK2-1C酿酒酵母进行构建，第一种构建方式获得的能够合成酪醇的酿酒酵母，其酪醇发酵产量为1040.2mg/L，第二种构建方式获得的能够合成酪醇的酿酒酵母，其酪醇发酵产量可高达1350mg/L，第三种构建方式在第二种方式基础上不过表达来源于大肠杆菌的TyrA^{M53I V354V}双突变体，酪醇发酵产量为1160mg/L；

其中，ARO2氨基酸序列如SEQ ID No.11所示，核苷酸序列如SEQ ID No.12所示；ARO10氨基酸序列如SEQ ID No.13所示，核苷酸序列如SEQ ID No.14所示；TKL1氨基酸序列如SEQ ID No.15所示，核苷酸序列如SEQ ID No.16所示；RKI1氨基酸序列如SEQ ID No.17所示，核苷酸序列如SEQ ID No.18所示；

ARO4^K229L氨基酸序列如SEQ ID No.19所示，核苷酸序列如SEQ ID No.20所示；ARO7^G141S氨基酸序列如SEQ ID No.21所示，核苷酸序列如SEQ ID No.22所示；ARO3^D154N突变体氨基酸序列如SEQ ID No.23所示，核苷酸序列如SEQ ID No.24所示；

来源于大肠杆菌的TyrA^{M53I V354V}双突变体氨基酸序列如SEQ ID No.25所示，核苷酸序列如SEQ ID No.26所示。

此外，为了进一步提高本发明所述高产羟基酪醇的酿酒酵母的产量，其内源的TRP2基因启动子(也可以说是所述能够合成酪醇的酿酒酵母的内源的TRP2基因启动子)可以更换为弱启动子，所述弱启动子为启动能力弱于原始启动子的启动子。在本发明具体实施方式中，本发明以YEN1p弱启动子替换TRP2基因的原始较强的启动子TRP2-promoter。

在进行了上述不同组合方案改造之后，本发明所述高产羟基酪醇的酿酒酵母的产量最高可达到1120mg/L。基于上述各项的技术效果，本发明提出了所述高产羟基酪醇的酿酒酵母在制备羟基酪醇中的应用；本发明还提出了如下组合的HpaB和HpaC或它们的表达载体以能够合成酪醇的酿酒酵母为底盘在构建高产羟基酪醇的酿酒酵母中的应用：

(1)来源于铜绿假单胞菌的HpaB，以及来源于肠沙门氏菌、铜绿假单胞菌或大肠杆菌的HpaC；或

(2)来源于大肠杆菌的HpaB和来源于铜绿假单胞菌的HpaC。

同时，本发明还提供了所述高产羟基酪醇的酿酒酵母的构建方法，将表达如下任意组合的HpaB和HpaC的重组载体转入能够合成酪醇的酿酒酵母中，获得所述高产羟基酪醇的酿酒酵母；

或将如下任意组合的HpaB和HpaC的编码基因整合到能够合成酪醇的酿酒酵母基因组中进行表达，获得所述高产羟基酪醇的酿酒酵母；

(2)来源于大肠杆菌的HpaB和来源于铜绿假单胞菌的HpaC。

所述重组载体亦可以称为表达盒，其是本领域公知的能够表达目标基因的基因原件，其上包括但不限于启动子、目标基因和终止子，这些根据实际需求可通过市售途径获得；在本发明具体实施方式中，所述重组载体以市售的PRS系列质粒(PRS403、PRS404、PRS406等)为基础进行构建；表达HpaB和HpaC的编码基因可以分别整合到载体质粒上形成不同的重组载体，也可以同时整合到相同的载体质粒上，这样可以更加方便和高效。

由于酿酒酵母特有的同源重组机制，本发明可以在HpaB和HpaC的编码基因两端构建同源臂，然后整合到酿酒酵母的基因组中。

为了能够进一步提高产量，优选地，采用前述构建方法获得能够合成酪醇的酿酒酵母；也可以对其内源的TRP2基因的启动子更换为启动能力弱于原始启动子的启动子。在本发明具体实施方式中，通过CRISPR-Cas9技术进行原始启动子的替换。

根据本发明提供的应用，还提供了一种制备羟基酪醇的方法，采用所述高产羟基酪醇的酿酒酵母进行发酵培养，收集发酵液上清分离出羟基酪醇。其中，所述发酵培养基优选为YPD培养基。

在本发明具体实施方式中，所述制备羟基酪醇的方法包括：

1)在YPD平板培养高产羟基酪醇的酿酒酵母菌株，活化酿酒酵母菌株；

2)挑取单克隆于5ml YPD液体，培养36小时；

3)将5ml YPD培养液按照1:50转接至50ml YPD，在30℃，220rpm培养84小时；

4)收集发酵液上清并进行分离，即可获得羟基酪醇。

由以上技术方案可知，本发明主要选择来源于铜绿假单胞菌的4-羟基苯乙酸-3-羟化酶，并结合其他特定来源的核黄素氧化还原酶，将其转入到能够生产酪醇的酿酒酵母底盘细胞中，实现了羟基酪醇产量的提高；在此基础上进一步进行多种改造，使得所述酿酒酵母的羟基酪醇的产量高达1120mg/L，为微生物高效发酵生产羟基酪醇提供了新的途径。

附图说明

图1所示为不同HpaB/C组合的产羟基酪醇酿酒酵母的产量结果；

图2所示为使用PaHpaB^Q212D突变体和EcHpaC组合的酿酒酵母羟基酪醇的产量结果；

图3所示为使用PaHpaB^Q212D突变体+EcHpaC组合的酿酒酵母，将TRP2基因原始启动子替换为YEN1p启动子前后的羟基酪醇的产量结果。

具体实施方式

本发明实施例公开了一种高产羟基酪醇的酿酒酵母及其构建方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明所述高产羟基酪醇的酿酒酵母及其构建方法已通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的高产羟基酪醇的酿酒酵母及其构建方法进行改动或适当变更与组合，来实现和应用本发明技术。

本发明在具体实施例中对比实验，除去各组设置的应有区别外，其他未明确提及的实验条件、原料、菌株、试剂等均保持一致，确保对比试验的可对比性。

以下就本发明所提供的一种高产羟基酪醇的酿酒酵母及其构建方法做进一步说明。

实施例1：产酪醇的底盘菌株的构建

1)以酿酒酵母CEN.PK2-1C基因组为模板，利用PCR扩增ARO2，ARO10，TKL1，RKI1基因片段和ARO4，ARO7，ARO3片段；以大肠杆菌BL21基因组为模板，利用PCR扩增TyrA片段；

2)利用点突变的方法获得ARO4^K229L，ARO7^G141S，ARO3^D154N突变体；

3)利用酿酒酵母组成型启动子表达上述基因，并构建至PRS406和PRS404整合质粒；

4)在上述酿酒酵母中利用CRISPR-Cas9的方法敲除PDC1和PHA2基因；

PDC1-spacer：ATTGGATCTGACTCCTCACG；

PDC1同源臂：

ACAGCAGCAAAATGACGATAGTTCCATAAATATGTATCCCGTGTATGCGTATTTGCCATCCATATCTAAAATTGGCACAATTGAACAACCCTGATAGAAAGGAATCATTTCTGTTGGAAA；

PHA2-spacer：GGGGGATAGAGGCTGCTGGG；

PHA2同源臂：

AGGTACGTATTCCCATCAAGCTGCATTACAACAATTTCAATCAACATCTGATGTTGAGTAAATGTTTTAACCAATTGGAGAACGACACTAGTATAGATTATTCAGTGGTACCGTTGG；

5)利用点突变的方法获得TyrA^{M53I V354V}双突变体基因，并构建至PRS405整合质粒。

6)将上述整合质粒整合至酿酒酵母CEN.PK2-1C中，获得高产酪醇的底盘酿酒酵母，将其用YPD培养基进行发酵，产量为1160mg/L；同时，本实施例也测试了不过表达TyrA^{M53I V354V}双突变体的底盘酿酒酵母的酪醇产量，HPLC检测结果为1350mg/L。此外，本发明也测试了不过表达ARO3^D154N突变体以及TyrA^{M53I V354V}双突变体的底盘酿酒酵母的酪醇产量，HPLC检测结果为1040.2mg/L；

实施例2：构建HpaB与HpaC组合

1)由试剂公司合成不同来源的HpaB与HpaC的编码基因，并以此为模板设计上下游引物(扩增引物两端添加PDC1p启动子同源臂和GPM1t终止子同源臂的序列，以及添加TEF1p启动子同源臂和PGK1t终止子同源臂的序列)进行PCR扩增。PCR扩增条件如下：

先95℃预变性10min，然后98℃30s，58℃30s，72℃1min 30s，共25个循环；最后72℃延伸5min。

胶回收上述PCR产物，获得带有PDC1p启动子同源臂和GPM1t终止子同源臂的不同来源的HpaB编码基因片段，以及带有TEF1p启动子同源臂和PGK1t终止子同源臂的HpaC编码基因片段。

2)将上述带有PDC1p启动子同源臂和GPM1t终止子同源臂的HpaB基因利用同源重组连接在带有PDC1p启动子和GPM1t终止子的载体PRS403-PDC1p-GPM1t，构建成能够在酿酒酵母菌株表达的HpaB表达盒；将上述带有TEF1p启动子同源臂和PGK1t终止子同源臂的HpaC基因利用同源重组连接在带有TEF1p启动子和PGK1t终止子的载体PRS403-TEF1p-PGK1t，构建能够在酿酒酵母菌株中表达HpaC表达盒(重组表达载体)。

3)体外利用PCR扩增不同的HpaC表达盒(带启动子和终止子)，连接至HpaB的表达盒，构建HpaB/C不同组合的共表达盒；

4)将上述不同HpaB/C组合的共表达盒导入至产酪醇的酿酒酵母菌株中，经测序验证，成功整合至酿酒酵母基因组，获得所述高产羟基酪醇的酿酒酵母。

实施例3：构建表达PaHpaB^Q212D突变体酿酒酵母

1)利用CRISPR-Cas9对实施例2中构建的酿酒酵母菌株突变PaHpaB为PaHpaB^Q212D。设计spacer为5’-TCAAGGTTCTGCTCAATTGT-3’，同源臂序列为：

TTGTTTCTGGTGCTAAGGTTGTTGCTACTAACTCTGCTTTGACTCACTACAACTTCGTTGGTCAAGGTTCTGCTGACCTACTTGGTGACAACACTGACTTCGCTTTGATGTTCATCGCTCCAATGAACACTCCAGGTAT；

2)将带有相应spacer的Cas9质粒和同源臂转入表达PaHpaB-HpaC组合的酿酒酵母菌株，经过菌落PCR和基因组PCR扩增PaHpaB序列后，测序验证PaHpaB成功突变为PaHpaB^Q212D。

实施例4：弱表达TRP2基因酿酒酵母菌株的构建

在实施例2构建的菌株或实施例3构建的菌株基础上，进行如下步骤：

1)利用CRISPR-Cas9构建弱表达TRP2基因，用弱启动子替换TRP2原始启动子，优选地，用YEN1p弱启动子替换TRP2原始启动子。设计spacer为5’-CTATGGTCCGTCGTAGCAGA-3’，SEQ ID No.27所示为带有同源臂的YEN1p弱启动子序列，其中489bp-916bp为YEN1p弱启动子序列，其余为同源臂序列，可根据实际情形调整；

2)将带有相应spacer的Cas9质粒和同源臂转入酿酒酵母菌株，经过菌落PCR和基因组PCR扩增后，经测序验证正确替换TRP2原始启动子。

实施例5：制备羟基酪醇的试验

1、发酵方法

2)挑取单克隆于5ml YPD液体，培养36小时；

4)收集发酵液上清，即可获得含有羟基酪醇的发酵液；HPLC检测羟基酪醇的产量。

2、试验结果

(1)按照实施例1的方法构建不同HpaB/C组合的产羟基酪醇酿酒酵母，其中，产酪醇的酿酒酵母各组均相同，参照实施例1的技术方法按照如下方式构建，同时各实验条件均保持一致：

过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L和ARO7^G141S突变体，以及敲除酿酒酵母PHA2和PDC1的表达；

然后构建的产羟基酪醇酿酒酵母进行发酵实验，结果见图1；

图1结果显示，相比较EcHpaB+SeHpaC组合和EcHpaB+EcHpaC组合，PaHpaB+PaHpaC、PaHpaB+EcHpaC、PaHpaB+SeHpaC和EcHpaB+PaHpaC组合均有显著性的产量提升，其中以PaHpaB+EcHpaC效果最佳，这说明不同HpaB/C组合的引入直接影响酿酒酵母的羟基酪醇产量，而产量高低并不能提前预期。

(2)按照实施例2的方法构建PaHpaB^Q212D突变体酿酒酵母，HpaB/C组合为(1)中最佳的PaHpaB+EcHpaC，产酪醇的酿酒酵母同(1)中底盘菌株，发酵实验结果见图2；图2结果显示采用PaHpaB^Q212D突变体可以进一步提高菌株的羟基酪醇产量。

(3)在(2)中菌株基础上按照实施例3方法构建弱表达TRP2基因酿酒酵母菌株，发酵实验结果见图3；图3结果显示YEN1p弱表达TRP2基因可以再次提高菌株的羟基酪醇产量。

(4)参照实施例1的技术方法按照如下方式构建产酪醇的酿酒酵母：

过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L、ARO7^G141S和ARO3^D154N突变体，过表达来源于大肠杆菌的TyrA^{M53I V354V}双突变体，以及敲除酿酒酵母PHA2和PDC1的表达；

然后根据(1)和(3)结果采用PaHpaB+EcHpaC组合，并且采用弱启动子YEN1p表达TRP2基因，所获的的菌株发酵后产量高达1120mg/L。

以上所述只是用于理解本发明的方法及其核心思想，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利的保护范围。

序列表

<110> 天津大学

<120> 一种高产羟基酪醇的酿酒酵母及其构建方法

<130> MP21007301

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 520

<212> PRT

<213> 铜绿假单胞菌(Pseudomonas aeruginosa)

<400> 1

Met Lys Pro Glu Asp Phe Arg Ala Ser Ala Thr Arg Pro Phe Thr Gly

1 5 10 15

Glu Glu Tyr Leu Ala Ser Leu Arg Asp Asp Arg Glu Ile Tyr Ile Tyr

20 25 30

Gly Asp Arg Val Lys Asp Val Thr Ser His Pro Ala Phe Arg Asn Ala

35 40 45

Ala Ala Ser Met Ala Arg Leu Tyr Asp Ala Leu His Asp Pro Gln Ser

50 55 60

Lys Glu Lys Leu Cys Trp Glu Thr Asp Thr Gly Asn Gly Gly Tyr Thr

65 70 75 80

His Lys Phe Phe Arg Tyr Ala Arg Ser Ala Asp Glu Leu Arg Gln Gln

85 90 95

Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Thr Tyr Gly Trp Met Gly

100 105 110

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

115 120 125

Pro Gly Phe Tyr Gly Arg Phe Glu Asp Asn Ala Lys Thr Trp Tyr Lys

130 135 140

Arg Ile Gln Glu Ala Cys Leu Tyr Leu Asn His Ala Ile Val Asn Pro

145 150 155 160

Pro Ile Asp Arg Asp Lys Pro Val Asp Gln Val Lys Asp Val Phe Ile

165 170 175

Ser Val Asp Glu Glu Val Asp Gly Gly Ile Val Val Ser Gly Ala Lys

180 185 190

Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Phe Val Gly Gln

195 200 205

Gly Ser Ala Gln Leu Leu Gly Asp Asn Thr Asp Phe Ala Leu Met Phe

210 215 220

Ile Ala Pro Met Asn Thr Pro Gly Met Lys Leu Ile Cys Arg Pro Ser

225 230 235 240

Tyr Glu Leu Val Ala Gly Ile Ala Gly Ser Pro Phe Asp Tyr Pro Leu

245 250 255

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

260 265 270

Phe Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Glu Arg Cys

275 280 285

Lys Gln Trp Phe Pro Gln Gly Gly Phe Gly Arg Leu Phe Pro Met Gln

290 295 300

Gly Cys Thr Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Gly Ala Leu

305 310 315 320

Tyr Lys Ala Leu Gln Cys Thr Gly Ser Leu Glu Phe Arg Gly Val Gln

325 330 335

Ala Gln Val Gly Glu Val Val Ala Trp Arg Asn Leu Phe Trp Ser Leu

340 345 350

Thr Asp Ala Met Tyr Gly Asn Ala Ser Glu Trp His Gly Gly Ala Phe

355 360 365

Leu Pro Ser Ala Glu Ala Leu Gln Ala Tyr Arg Val Leu Ala Pro Gln

370 375 380

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

385 390 395 400

Leu Ile Tyr Leu Pro Ser Gly Val Arg Asp Leu His Asn Pro Gln Leu

405 410 415

Asp Lys Tyr Leu Ser Thr Tyr Cys Arg Gly Ser Gly Gly Met Gly His

420 425 430

Arg Glu Arg Ile Lys Ile Leu Lys Leu Leu Trp Asp Ala Ile Gly Ser

435 440 445

Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ala Gly Ser

450 455 460

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

465 470 475 480

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

485 490 495

Asp Glu Asn Gly Trp Thr Val Pro His Leu His Asn Pro Asp Asp Ile

500 505 510

Asn Val Leu Asp Arg Ile Arg Gln

515 520

<210> 2

<211> 1563

<212> DNA

<213> 铜绿假单胞菌(Pseudomonas aeruginosa)

<400> 2

atgaagccag aagacttcag agcttctgct actagaccat tcactggtga agaatacttg 60

gcttctttga gagacgacag agaaatctac atctacggtg acagagttaa ggacgttact 120

tctcacccag ctttcagaaa cgctgctgct tctatggcta gattgtacga cgctttgcac 180

gacccacaat ctaaggaaaa gttgtgttgg gaaactgaca ctggtaacgg tggttacact 240

cacaagttct tcagatacgc tagatctgct gacgaattga gacaacaaag agacgctatc 300

gctgaatggt ctagattgac ttacggttgg atgggtagaa ctccagacta caaggctgct 360

ttcggttctg ctttgggtgc taacccaggt ttctacggta gattcgaaga caacgctaag 420

acttggtaca agagaatcca agaagcttgt ttgtacttga accacgctat cgttaaccca 480

ccaatcgaca gagacaagcc agttgaccaa gttaaggacg ttttcatctc tgttgacgaa 540

gaagttgacg gtggtatcgt tgtttctggt gctaaggttg ttgctactaa ctctgctttg 600

actcactaca acttcgttgg tcaaggttct gctcaattgt tgggtgacaa cactgacttc 660

gctttgatgt tcatcgctcc aatgaacact ccaggtatga agttgatctg tagaccatct 720

tacgaattgg ttgctggtat cgctggttct ccattcgact acccattgtc ttctagattc 780

gacgaaaacg acgctatctt ggttatggac aaggttttca tcccatggga aaacgttttg 840

atctacagag acttcgaaag atgtaagcaa tggttcccac aaggtggttt cggtagattg 900

ttcccaatgc aaggttgtac tagattggct gttaagttgg acttcatcac tggtgctttg 960

tacaaggctt tgcaatgtac tggttctttg gaattcagag gtgttcaagc tcaagttggt 1020

gaagttgttg cttggagaaa cttgttctgg tctttgactg acgctatgta cggtaacgct 1080

tctgaatggc acggtggtgc tttcttgcca tctgctgaag ctttgcaagc ttacagagtt 1140

ttggctccac aagcttaccc agaaatcaag aagactatcg aacaagttgt tgcttctggt 1200

ttgatctact tgccatctgg tgttagagac ttgcacaacc cacaattgga caagtacttg 1260

tctacttact gtagaggttc tggtggtatg ggtcacagag aaagaatcaa gatcttgaag 1320

ttgttgtggg acgctatcgg ttctgaattc ggtggtagac acgaattgta cgaaatcaac 1380

tacgctggtt ctcaagacga aatcagaatg caagctttga gacaagctat cggttctggt 1440

gctatgaagg gtatgttggg tatggttgaa caatgtatgg gtgactacga cgaaaacggt 1500

tggactgttc cacacttgca caacccagac gacatcaacg ttttggacag aatcagacaa 1560

taa 1563

<210> 3

<211> 170

<212> PRT

<213> 肠沙门氏菌(Salmonella enterica)

<400> 3

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

1 5 10 15

Leu Ala Ala Ala Val Asn Ile Val Thr Thr Ala Gly His Ala Gly Arg

20 25 30

Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Thr Pro Pro

35 40 45

Ser Val Met Val Cys Ile Asn Ala Asn Ser Ala Met Asn Pro Val Phe

50 55 60

Gln Gly Asn Gly Arg Leu Cys Ile Asn Val Leu Asn His Glu Gln Glu

65 70 75 80

Leu Met Ala Arg His Phe Ala Gly Met Thr Gly Met Ala Met Glu Glu

85 90 95

Arg Phe His Gln Pro Cys Trp Gln Asn Gly Pro Leu Gly Gln Pro Val

100 105 110

Leu Asn Gly Ala Leu Ala Gly Leu Glu Gly Glu Ile Ser Glu Val Gln

115 120 125

Thr Ile Gly Thr His Leu Val Tyr Leu Val Ala Ile Lys Asn Ile Ile

130 135 140

Leu Ser Gln Asp Gly His Gly Leu Ile Tyr Phe Lys Arg Arg Phe His

145 150 155 160

Pro Val Arg Leu Glu Met Glu Ala Pro Val

165 170

<210> 4

<211> 170

<212> PRT

<213> 铜绿假单胞菌(Pseudomonas aeruginosa)

<400> 4

Met Ser Gln Leu Glu Pro Arg Gln Gln Ala Phe Arg Asn Ala Met Ala

1 5 10 15

His Leu Ser Ala Ala Val Asn Val Ile Thr Ser Asn Gly Pro Ala Gly

20 25 30

Arg Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Ser Pro

35 40 45

Pro Thr Leu Met Leu Cys Ile Asn Arg Asn Ser Glu Met Asn Thr Val

50 55 60

Phe Lys Ala Asn Gly Arg Leu Cys Val Asn Val Leu Ser Gly Glu His

65 70 75 80

Glu Glu Val Ala Arg His Phe Ala Gly Met Thr Glu Val Pro Met Glu

85 90 95

Arg Arg Phe Ala Leu His Asp Trp Arg Glu Gly Leu Ala Gly Leu Pro

100 105 110

Val Leu His Gly Ala Leu Ala Asn Leu Gln Gly Arg Ile Ala Glu Val

115 120 125

Gln Glu Ile Gly Thr His Ser Val Leu Leu Leu Glu Leu Glu Asp Ile

130 135 140

Gln Val Leu Glu Gln Gly Asp Gly Leu Val Tyr Phe Ser Arg Ser Phe

145 150 155 160

His Arg Leu Gln Cys Pro Arg Arg Ala Ala

165 170

<210> 5

<211> 170

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 5

Met Gln Leu Asp Glu Gln Arg Leu Arg Phe Arg Asp Ala Met Ala Ser

1 5 10 15

Leu Ser Ala Ala Val Asn Ile Ile Thr Thr Glu Gly Asp Ala Gly Gln

20 25 30

Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Thr Pro Pro

35 40 45

Ser Leu Met Val Cys Ile Asn Ala Asn Ser Ala Met Asn Pro Val Phe

50 55 60

Gln Gly Asn Gly Lys Leu Cys Val Asn Val Leu Asn His Glu Gln Glu

65 70 75 80

Leu Met Ala Arg His Phe Ala Gly Met Thr Gly Met Ala Met Glu Glu

85 90 95

Arg Phe Ser Leu Ser Cys Trp Gln Lys Gly Pro Leu Ala Gln Pro Val

100 105 110

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

115 120 125

Ala Ile Gly Thr His Leu Val Tyr Leu Val Glu Ile Lys Asn Ile Ile

130 135 140

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

145 150 155 160

Pro Val Met Leu Glu Met Glu Ala Ala Ile

165 170

<210> 6

<211> 513

<212> DNA

<213> 肠沙门氏菌(Salmonella enterica)

<400> 6

atgcaagttg acgaacaaag attgcacttc agagacgcta tggcttcttt ggctgctgct 60

gttaacatcg ttactactgc tggtcacgct ggtagatgtg gtatcactgc tactgctgtt 120

tgttctgtta ctgacactcc accatctgtt atggtttgta tcaacgctaa ctctgctatg 180

aacccagttt tccaaggtaa cggtagattg tgtatcaacg ttttgaacca cgaacaagaa 240

ttgatggcta gacacttcgc tggtatgact ggtatggcta tggaagaaag attccaccaa 300

ccatgttggc aaaacggtcc attgggtcaa ccagttttga acggtgcttt ggctggtttg 360

gaaggtgaaa tctctgaagt tcaaactatc ggtactcact tggtttactt ggttgctatc 420

aagaacatca tcttgtctca agacggtcac ggtttgatct acttcaagag aagattccac 480

ccagttagat tggaaatgga agctccagtt taa 513

<210> 7

<211> 513

<212> DNA

<213> 铜绿假单胞菌(Pseudomonas aeruginosa)

<400> 7

atgtctcaat tggaaccaag acaacaagct ttcagaaacg ctatggctca cttgtctgct 60

gctgttaacg ttatcacttc taacggtcca gctggtagat gtggtatcac tgctactgct 120

gtttgttctg ttactgactc tccaccaact ttgatgttgt gtatcaacag aaactctgaa 180

atgaacactg ttttcaaggc taacggtaga ttgtgtgtta acgttttgtc tggtgaacac 240

gaagaagttg ctagacactt cgctggtatg actgaagttc caatggaaag aagattcgct 300

ttgcacgact ggagagaagg tttggctggt ttgccagttt tgcacggtgc tttggctaac 360

ttgcaaggta gaatcgctga agttcaagaa atcggtactc actctgtttt gttgttggaa 420

ttggaagaca tccaagtttt ggaacaaggt gacggtttgg tttacttctc tagatctttc 480

cacagattgc aatgtccaag aagagctgct taa 513

<210> 8

<211> 513

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<400> 8

atgcaattgg acgaacaaag attgagattc agagacgcta tggcttcttt gtctgctgct 60

gttaacatca tcactactga aggtgacgct ggtcaatgtg gtatcactgc tactgctgtt 120

tgttctgtta ctgacactcc accatctttg atggtttgta tcaacgctaa ctctgctatg 180

aacccagttt tccaaggtaa cggtaagttg tgtgttaacg ttttgaacca cgaacaagaa 240

ttgatggcta gacacttcgc tggtatgact ggtatggcta tggaagaaag attctctttg 300

tcttgttggc aaaagggtcc attggctcaa ccagttttga agggttcttt ggcttctttg 360

gaaggtgaaa tcagagacgt tcaagctatc ggtactcact tggtttactt ggttgaaatc 420

aagaacatca tcttgtctgc tgaaggtcac ggtttgatct acttcaagag aagattccac 480

ccagttatgt tggaaatgga agctgctatc taa 513

<210> 9

<211> 520

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 9

Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly

1 5 10 15

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

20 25 30

Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala

35 40 45

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

50 55 60

Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr

65 70 75 80

His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg Gln Gln

85 90 95

Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly

100 105 110

Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Ala Asn

115 120 125

Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr

130 135 140

Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro

145 150 155 160

Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile

165 170 175

Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys

180 185 190

Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe

195 200 205

Gly Ser Ala Gln Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe

210 215 220

Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser

225 230 235 240

Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu

245 250 255

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

260 265 270

Leu Ile Pro Trp Glu Asn Val Leu Ile Tyr Arg Asp Phe Asp Arg Cys

275 280 285

Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln

290 295 300

Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu

305 310 315 320

Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln

325 330 335

Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu

340 345 350

Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr

355 360 365

Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met

370 375 380

Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly

385 390 395 400

Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile

405 410 415

Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His

420 425 430

Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser

435 440 445

Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser

450 455 460

Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Asn Ser Gly

465 470 475 480

Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr

485 490 495

Asp Gln Asp Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile

500 505 510

Asn Met Leu Asp Lys Leu Leu Lys

515 520

<210> 10

<211> 1563

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<400> 10

atgaagccag aagacttcag agcttctact caaagaccat tcactggtga agaatacttg 60

aagtctttgc aagacggtag agaaatctac atctacggtg aaagagttaa ggacgttact 120

actcacccag ctttcagaaa cgctgctgct tctgttgctc aattgtacga cgctttgcac 180

aagccagaaa tgcaagactc tttgtgttgg aacactgaca ctggttctgg tggttacact 240

cacaagttct tcagagttgc taagtctgct gacgacttga gacaacaaag agacgctatc 300

gctgaatggt ctagattgtc ttacggttgg atgggtagaa ctccagacta caaggctgct 360

ttcggttgtg ctttgggtgc taacccaggt ttctacggtc aattcgaaca aaacgctaga 420

aactggtaca ctagaatcca agaaactggt ttgtacttca accacgctat cgttaaccca 480

ccaatcgaca gacacttgcc aactgacaag gttaaggacg tttacatcaa gttggaaaag 540

gaaactgacg ctggtatcat cgtttctggt gctaaggttg ttgctactaa ctctgctttg 600

actcactaca acatgatcgg ttttggctct gctcaagtca tgggtgaaaa cccagacttc 660

gctttgatgt tcgttgctcc aatggacgct gacggtgtta agttgatctc tagagcttct 720

tacgaaatgg ttgctggtgc tactggttct ccatacgact acccattgtc ttctagattc 780

gacgaaaacg acgctatctt ggttatggac aacgttttga tcccatggga aaacgttttg 840

atctacagag acttcgacag atgtagaaga tggactatgg aaggtggttt cgctagaatg 900

tacccattgc aagcttgtgt tagattggct gttaagttgg acttcatcac tgctttgttg 960

aagaagtctt tggaatgtac tggtactttg gaattcagag gtgttcaagc tgacttgggt 1020

gaagttgttg cttggagaaa cactttctgg gctttgtctg actctatgtg ttctgaagct 1080

actccatggg ttaacggtgc ttacttgcca gaccacgctg ctttgcaaac ttacagagtt 1140

ttggctccaa tggcttacgc taagatcaag aacatcatcg aaagaaacgt tacttctggt 1200

ttgatctact tgccatcttc tgctagagac ttgaacaacc cacaaatcga ccaatacttg 1260

gctaagtacg ttagaggttc taacggtatg gaccacgttc aaagaatcaa gatcttgaag 1320

ttgatgtggg acgctatcgg ttctgaattc ggtggtagac acgaattgta cgaaatcaac 1380

tactctggtt ctcaagacga aatcagattg caatgtttga gacaagctca aaactctggt 1440

aacatggaca agatgatggc tatggttgac agatgtttgt ctgaatacga ccaagacggt 1500

tggactgttc cacacttgca caacaacgac gacatcaaca tgttggacaa gttgttgaag 1560

taa 1563

<210> 11

<211> 376

<212> PRT

<213> 酿酒酵母(CEN.PK2-1C)

<400> 11

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

1 5 10 15

His Cys Lys Ser Val Gly Cys Ile Val Asp Gly Val Pro Pro Gly Met

20 25 30

Ser Leu Thr Glu Ala Asp Ile Gln Pro Gln Leu Thr Arg Arg Arg Pro

35 40 45

Gly Gln Ser Lys Leu Ser Thr Pro Arg Asp Glu Lys Asp Arg Val Glu

50 55 60

Ile Gln Ser Gly Thr Glu Phe Gly Lys Thr Leu Gly Thr Pro Ile Ala

65 70 75 80

Met Met Ile Lys Asn Glu Asp Gln Arg Pro His Asp Tyr Ser Asp Met

85 90 95

Asp Lys Phe Pro Arg Pro Ser His Ala Asp Phe Thr Tyr Ser Glu Lys

100 105 110

Tyr Gly Ile Lys Ala Ser Ser Gly Gly Gly Arg Ala Ser Ala Arg Glu

115 120 125

Thr Ile Gly Arg Val Ala Ser Gly Ala Ile Ala Glu Lys Phe Leu Ala

130 135 140

Gln Asn Ser Asn Val Glu Ile Val Ala Phe Val Thr Gln Ile Gly Glu

145 150 155 160

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

165 170 175

Asn Thr Ile Thr Arg Glu Lys Val Asp Ser Met Gly Pro Ile Arg Cys

180 185 190

Pro Asp Ala Ser Val Ala Gly Leu Met Val Lys Glu Ile Glu Lys Tyr

195 200 205

Arg Gly Asn Lys Asp Ser Ile Gly Gly Val Val Thr Cys Val Val Arg

210 215 220

Asn Leu Pro Thr Gly Leu Gly Glu Pro Cys Phe Asp Lys Leu Glu Ala

225 230 235 240

Met Leu Ala His Ala Met Leu Ser Ile Pro Ala Ser Lys Gly Phe Glu

245 250 255

Ile Gly Ser Gly Phe Gln Gly Val Ser Val Pro Gly Ser Lys His Asn

260 265 270

Asp Pro Phe Tyr Phe Glu Lys Glu Thr Asn Arg Leu Arg Thr Lys Thr

275 280 285

Asn Asn Ser Gly Gly Val Gln Gly Gly Ile Ser Asn Gly Glu Asn Ile

290 295 300

Tyr Phe Ser Val Pro Phe Lys Ser Val Ala Thr Ile Ser Gln Glu Gln

305 310 315 320

Lys Thr Ala Thr Tyr Asp Gly Glu Glu Gly Ile Leu Ala Ala Lys Gly

325 330 335

Arg His Asp Pro Ala Val Thr Pro Arg Ala Ile Pro Ile Val Glu Ala

340 345 350

Met Thr Ala Leu Val Leu Ala Asp Ala Leu Leu Ile Gln Lys Ala Arg

355 360 365

Asp Phe Ser Arg Ser Val Val His

370 375

<210> 12

<211> 1131

<212> DNA

<213> 酿酒酵母(CEN.PK2-1C)

<400> 12

atgtcaacgt ttgggaaact gttccgcgtc accacatatg gtgaatcgca ttgtaagtct 60

gtcggttgca ttgtcgacgg tgttcctcca ggaatgtcat taaccgaagc tgacattcag 120

ccacaattga ccagaagaag accgggtcaa tctaagctat cgacccctag agacgaaaag 180

gatagagtgg aaatccagtc cggtaccgag ttcggcaaga ctctaggtac acccatcgcc 240

atgatgatca aaaacgagga ccaaagacct cacgactact ccgacatgga caagttccct 300

agaccttccc atgcggactt cacgtactcg gaaaagtacg gtatcaaggc ctcctctggt 360

ggtggcagag cttctgctag agaaacgatt ggccgtgtcg cttcaggtgc cattgctgag 420

aagttcttag ctcagaactc taatgtcgag atcgtagcct ttgtgacaca aatcggggaa 480

atcaagatga acagagactc tttcgatcct gaatttcagc atctgttgaa caccatcacc 540

agggaaaaag tggactcaat gggtcctatc agatgtccag acgcctccgt tgctggtttg 600

atggtcaagg aaatcgaaaa gtacagaggc aacaaggact ctatcggtgg tgtcgtcact 660

tgtgtcgtga gaaacttgcc taccggtctc ggtgagccat gctttgacaa gttggaagcc 720

atgttggctc atgctatgtt gtccattcca gcatccaagg gtttcgaaat tggctcaggt 780

tttcagggtg tctctgttcc agggtccaag cacaatgacc cattttactt tgaaaaagaa 840

acaaacagat taagaacaaa gaccaacaat tcaggtggtg tacaaggtgg tatctctaat 900

ggtgagaaca tctatttctc tgtcccattc aagtcagtgg ccactatctc tcaagaacaa 960

aaaaccgcca cttacgatgg tgaagaaggt atcttagccg ctaagggtag acatgaccct 1020

gctgtcactc caagagctat tcctattgtg gaagccatga ccgctctggt gttggctgac 1080

gcgcttttga tccaaaaggc aagagatttc tccagatccg tggttcatta a 1131

<210> 13

<211> 635

<212> PRT

<213> 酿酒酵母(CEN.PK2-1C)

<400> 13

Met Ala Pro Val Thr Ile Glu Lys Phe Val Asn Gln Glu Glu Arg His

1 5 10 15

Leu Val Ser Asn Arg Ser Ala Thr Ile Pro Phe Gly Glu Tyr Ile Phe

20 25 30

Lys Arg Leu Leu Ser Ile Asp Thr Lys Ser Val Phe Gly Val Pro Gly

35 40 45

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

50 55 60

Ser Ala Gly Leu Arg Trp Val Gly Thr Cys Asn Glu Leu Asn Ala Ala

65 70 75 80

Tyr Ala Ala Asp Gly Tyr Ser Arg Tyr Ser Asn Lys Ile Gly Cys Leu

85 90 95

Ile Thr Thr Tyr Gly Val Gly Glu Leu Ser Ala Leu Asn Gly Ile Ala

100 105 110

Gly Ser Phe Ala Glu Asn Val Lys Val Leu His Ile Val Gly Val Ala

115 120 125

Lys Ser Ile Asp Ser Arg Ser Ser Asn Phe Ser Asp Arg Asn Leu His

130 135 140

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

145 150 155 160

Lys Val Tyr His Asp Met Val Lys Asp Arg Val Ala Cys Ser Val Ala

165 170 175

Tyr Leu Glu Asp Ile Glu Thr Ala Cys Asp Gln Val Asp Asn Val Ile

180 185 190

Arg Asp Ile Tyr Lys Tyr Ser Lys Pro Gly Tyr Ile Phe Val Pro Ala

195 200 205

Asp Phe Ala Asp Met Ser Val Thr Cys Asp Asn Leu Val Asn Val Pro

210 215 220

Arg Ile Ser Gln Gln Asp Cys Ile Val Tyr Pro Ser Glu Asn Gln Leu

225 230 235 240

Ser Asp Ile Ile Asn Lys Ile Thr Ser Trp Ile Tyr Ser Ser Lys Thr

245 250 255

Pro Ala Ile Leu Gly Asp Val Leu Thr Asp Arg Tyr Gly Val Ser Asn

260 265 270

Phe Leu Asn Lys Leu Ile Cys Lys Thr Gly Ile Trp Asn Phe Ser Thr

275 280 285

Val Met Gly Lys Ser Val Ile Asp Glu Ser Asn Pro Thr Tyr Met Gly

290 295 300

Gln Tyr Asn Gly Lys Glu Gly Leu Lys Gln Val Tyr Glu His Phe Glu

305 310 315 320

Leu Cys Asp Leu Val Leu His Phe Gly Val Asp Ile Asn Glu Ile Asn

325 330 335

Asn Gly His Tyr Thr Phe Thr Tyr Lys Pro Asn Ala Lys Ile Ile Gln

340 345 350

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

355 360 365

Gln Met Phe Lys Gly Ile Asn Phe Ala Pro Ile Leu Lys Glu Leu Tyr

370 375 380

Lys Arg Ile Asp Val Ser Lys Leu Ser Leu Gln Tyr Asp Ser Asn Val

385 390 395 400

Thr Gln Tyr Thr Asn Glu Thr Met Arg Leu Glu Asp Pro Thr Asn Gly

405 410 415

Gln Ser Ser Ile Ile Thr Gln Val His Leu Gln Lys Thr Met Pro Lys

420 425 430

Phe Leu Asn Pro Gly Asp Val Val Val Cys Glu Thr Gly Ser Phe Gln

435 440 445

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

450 455 460

Gln Gly Phe Phe Leu Ser Ile Gly Met Ala Leu Pro Ala Ala Leu Gly

465 470 475 480

Val Gly Ile Ala Met Gln Asp His Ser Asn Ala His Ile Asn Gly Gly

485 490 495

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

500 505 510

Gly Ala Ala Gln Met Thr Ile Gln Glu Leu Ser Thr Ile Leu Lys Cys

515 520 525

Asn Ile Pro Leu Glu Val Ile Ile Trp Asn Asn Asn Gly Tyr Thr Ile

530 535 540

Glu Arg Ala Ile Met Gly Pro Thr Arg Ser Tyr Asn Asp Val Met Ser

545 550 555 560

Trp Lys Trp Thr Lys Leu Phe Glu Ala Phe Gly Asp Phe Asp Gly Lys

565 570 575

Tyr Thr Asn Ser Thr Leu Ile Gln Cys Pro Ser Lys Leu Ala Leu Lys

580 585 590

Leu Glu Glu Leu Lys Asn Ser Asn Lys Arg Ser Gly Ile Glu Leu Leu

595 600 605

Glu Val Lys Leu Gly Glu Leu Asp Phe Pro Glu Gln Leu Lys Cys Met

610 615 620

Val Glu Ala Ala Ala Leu Lys Arg Asn Lys Lys

625 630 635

<210> 14

<211> 1908

<212> DNA

<213> 酿酒酵母(CEN.PK2-1C)

<400> 14

atggcacctg ttacaattga aaagttcgta aatcaagaag aacgacacct tgtttccaac 60

cgatcagcaa caattccgtt tggtgaatac atatttaaaa gattgttgtc catcgatacg 120

aaatcagttt tcggtgttcc tggtgacttc aacttatctc tattagaata tctctattca 180

cctagtgttg aatcagctgg cctaagatgg gtcggcacgt gtaatgaact gaacgccgct 240

tatgcggccg acggatattc ccgttactct aataagattg gctgtttaat aaccacgtat 300

ggcgttggtg aattaagcgc cttgaacggt atagccggtt cgttcgctga aaatgtcaaa 360

gttttgcaca ttgttggtgt ggccaagtcc atagattcgc gttcaagtaa ctttagtgat 420

cggaacctac atcatttggt cccacagcta catgattcaa attttaaagg gccaaatcat 480

aaagtatatc atgatatggt aaaagataga gtcgcttgct cggtagccta cttggaggat 540

attgaaactg catgtgacca agtcgataat gttatccgcg atatttacaa gtattctaaa 600

cctggttata tttttgttcc tgcagatttt gcggatatgt ctgttacatg tgataatttg 660

gttaatgttc cacgtatatc tcaacaagat tgtatagtat acccttctga aaaccaattg 720

tctgacataa tcaacaagat tactagttgg atatattcca gtaaaacacc tgcgatcctt 780

ggagacgtac tgactgatag gtatggtgtg agtaactttt tgaacaagct tatctgcaaa 840

actgggattt ggaatttttc cactgttatg ggaaaatctg taattgatga gtcaaaccca 900

acttatatgg gtcaatataa tggtaaagaa ggtttaaaac aagtctatga acattttgaa 960

ctgtgcgact tggtcttgca ttttggagtc gacatcaatg aaattaataa tgggcattat 1020

acttttactt ataaaccaaa tgctaaaatc attcaatttc atccgaatta tattcgcctt 1080

gtggacacta ggcagggcaa tgagcaaatg ttcaaaggaa tcaattttgc ccctatttta 1140

aaagaactat acaagcgcat tgacgtttct aaactttctt tgcaatatga ttcaaatgta 1200

actcaatata cgaacgaaac aatgcggtta gaagatccta ccaatggaca atcaagcatt 1260

attacacaag ttcacttaca aaagacgatg cctaaatttt tgaaccctgg tgatgttgtc 1320

gtttgtgaaa caggctcttt tcaattctct gttcgtgatt tcgcgtttcc ttcgcaatta 1380

aaatatatat cgcaaggatt tttcctttcc attggcatgg cccttcctgc cgccctaggt 1440

gttggaattg ccatgcaaga ccactcaaac gctcacatca atggtggcaa cgtaaaagag 1500

gactataagc caagattaat tttgtttgaa ggtgacggtg cagcacagat gacaatccaa 1560

gaactgagca ccattctgaa gtgcaatatt ccactagaag ttatcatttg gaacaataac 1620

ggctacacta ttgaaagagc catcatgggc cctaccaggt cgtataacga cgttatgtct 1680

tggaaatgga ccaaactatt tgaagcattc ggagacttcg acggaaagta tactaatagc 1740

actctcattc aatgtccctc taaattagca ctgaaattgg aggagcttaa gaattcaaac 1800

aaaagaagcg ggatagaact tttagaagtc aaattaggcg aattggattt ccccgaacag 1860

ctaaagtgca tggttgaagc agcggcactt aaaagaaata aaaaatag 1908

<210> 15

<211> 680

<212> PRT

<213> 酿酒酵母(CEN.PK2-1C)

<400> 15

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

1 5 10 15

Ile Leu Ala Val Asp Thr Val Ser Lys Ala Asn Ser Gly His Pro Gly

20 25 30

Ala Pro Leu Gly Met Ala Pro Ala Ala His Val Leu Trp Ser Gln Met

35 40 45

Arg Met Asn Pro Thr Asn Pro Asp Trp Ile Asn Arg Asp Arg Phe Val

50 55 60

Leu Ser Asn Gly His Ala Val Ala Leu Leu Tyr Ser Met Leu His Leu

65 70 75 80

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

85 90 95

Gly Ser Arg Thr Pro Gly His Pro Glu Phe Glu Leu Pro Gly Val Glu

100 105 110

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

115 120 125

Ala Met Ala Gln Ala Asn Leu Ala Ala Thr Tyr Asn Lys Pro Gly Phe

130 135 140

Thr Leu Ser Asp Asn Tyr Thr Tyr Val Phe Leu Gly Asp Gly Cys Leu

145 150 155 160

Gln Glu Gly Ile Ser Ser Glu Ala Ser Ser Leu Ala Gly His Leu Lys

165 170 175

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

180 185 190

Gly Ala Thr Ser Ile Ser Phe Asp Glu Asp Val Ala Lys Arg Tyr Glu

195 200 205

Ala Tyr Gly Trp Glu Val Leu Tyr Val Glu Asn Gly Asn Glu Asp Leu

210 215 220

Ala Gly Ile Ala Lys Ala Ile Ala Gln Ala Lys Leu Ser Lys Asp Lys

225 230 235 240

Pro Thr Leu Ile Lys Met Thr Thr Thr Ile Gly Tyr Gly Ser Leu His

245 250 255

Ala Gly Ser His Ser Val His Gly Ala Pro Leu Lys Ala Asp Asp Val

260 265 270

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

275 280 285

Val Pro Gln Glu Val Tyr Asp His Tyr Gln Lys Thr Ile Leu Lys Pro

290 295 300

Gly Val Glu Ala Asn Asn Lys Trp Asn Lys Leu Phe Ser Glu Tyr Gln

305 310 315 320

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

325 330 335

Gln Leu Pro Ala Asn Trp Glu Ser Lys Leu Pro Thr Tyr Thr Ala Lys

340 345 350

Asp Ser Ala Val Ala Thr Arg Lys Leu Ser Glu Thr Val Leu Glu Asp

355 360 365

Val Tyr Asn Gln Leu Pro Glu Leu Ile Gly Gly Ser Ala Asp Leu Thr

370 375 380

Pro Ser Asn Leu Thr Arg Trp Lys Glu Ala Leu Asp Phe Gln Pro Pro

385 390 395 400

Ser Ser Gly Ser Gly Asn Tyr Ser Gly Arg Tyr Ile Arg Tyr Gly Ile

405 410 415

Arg Glu His Ala Met Gly Ala Ile Met Asn Gly Ile Ser Ala Phe Gly

420 425 430

Ala Asn Tyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe Val Ser Tyr

435 440 445

Ala Ala Gly Ala Val Arg Leu Ser Ala Leu Ser Gly His Pro Val Ile

450 455 460

Trp Val Ala Thr His Asp Ser Ile Gly Val Gly Glu Asp Gly Pro Thr

465 470 475 480

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

485 490 495

Gln Val Trp Arg Pro Ala Asp Gly Asn Glu Val Ser Ala Ala Tyr Lys

500 505 510

Asn Ser Leu Glu Ser Lys His Thr Pro Ser Ile Ile Ala Leu Ser Arg

515 520 525

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

530 535 540

Gly Gly Tyr Val Leu Gln Asp Val Ala Asn Pro Asp Ile Ile Leu Val

545 550 555 560

Ala Thr Gly Ser Glu Val Ser Leu Ser Val Glu Ala Ala Lys Thr Leu

565 570 575

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

580 585 590

Thr Phe Asp Lys Gln Pro Leu Glu Tyr Arg Leu Ser Val Leu Pro Asp

595 600 605

Asn Val Pro Ile Met Ser Val Glu Val Leu Ala Thr Thr Cys Trp Gly

610 615 620

Lys Tyr Ala His Gln Ser Phe Gly Ile Asp Arg Phe Gly Ala Ser Gly

625 630 635 640

Lys Ala Pro Glu Val Phe Lys Phe Phe Gly Phe Thr Pro Glu Gly Val

645 650 655

Ala Glu Arg Ala Gln Lys Thr Ile Ala Phe Tyr Lys Gly Asp Lys Leu

660 665 670

Ile Ser Pro Leu Lys Lys Ala Phe

675 680

<210> 16

<211> 2043

<212> DNA

<213> 酿酒酵母(CEN.PK2-1C)

<400> 16

atgactcaat tcactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60

gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120

gcacacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180

gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240

actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300

ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360

ggtatctcca acgctgttgg tatggccatg gctcaagcta acctggctgc cacttacaac 420

aagccgggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480

caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540

attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600

gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660

aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720

ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780

tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840

ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900

attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960

aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020

aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080

ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140

gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200

tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag agaacacgct 1260

atgggtgcca taatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320

actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380

cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440

catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500

ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560

ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620

agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680

gctactggtt ccgaagtgtc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740

atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800

tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860

acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920

aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980

caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040

taa 2043

<210> 17

<211> 258

<212> PRT

<213> 酿酒酵母(CEN.PK2-1C)

<400> 17

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

1 5 10 15

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

20 25 30

Asn Leu Lys Phe Asp Asp His Lys Ile Ile Gly Ile Gly Ser Gly Ser

35 40 45

Thr Val Val Tyr Val Ala Glu Arg Ile Gly Gln Tyr Leu His Asp Pro

50 55 60

Lys Phe Tyr Glu Val Ala Ser Lys Phe Ile Cys Ile Pro Thr Gly Phe

65 70 75 80

Gln Ser Arg Asn Leu Ile Leu Asp Asn Lys Leu Gln Leu Gly Ser Ile

85 90 95

Glu Gln Tyr Pro Arg Ile Asp Ile Ala Phe Asp Gly Ala Asp Glu Val

100 105 110

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

115 120 125

Glu Lys Leu Val Ser Thr Ser Ala Lys Thr Phe Ile Val Val Ala Asp

130 135 140

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

145 150 155 160

Val Pro Ile Glu Ile Val Pro Ser Ser Tyr Val Arg Val Lys Asn Asp

165 170 175

Leu Leu Glu Gln Leu His Ala Glu Lys Val Asp Ile Arg Gln Gly Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Ser Lys Ala Tyr Phe Gly Asn Ser Asp Gly Ser Val Glu Val Thr

245 250 255

Glu Lys

<210> 18

<211> 777

<212> DNA

<213> 酿酒酵母(CEN.PK2-1C)

<400> 18

atggctgccg gtgtcccaaa aattgatgcg ttagaatctt tgggcaatcc tttggaggat 60

gccaagagag ctgcagcata cagagcagtt gatgaaaatt taaaatttga tgatcacaaa 120

attattggaa ttggtagtgg tagcacagtg gtttatgttg ccgaaagaat tggacaatat 180

ttgcatgacc ctaaatttta tgaagtagcg tctaaattca tttgcattcc aacaggattc 240

caatcaagaa acttgatttt ggataacaag ttgcaattag gctccattga acagtatcct 300

cgcattgata tagcgtttga cggtgctgat gaagtggatg agaatttaca attaattaaa 360

ggtggtggtg cttgtctatt tcaagaaaaa ttggttagta ctagtgctaa aaccttcatt 420

gtcgttgctg attcaagaaa aaagtcacca aaacatttag gtaagaactg gaggcaaggt 480

gttcccattg aaattgtacc ttcctcatac gtgagggtca agaatgatct attagaacaa 540

ttgcatgctg aaaaagttga catcagacaa ggaggttctg ctaaagcagg tcctgttgta 600

actgacaata ataacttcat tatcgatgcg gatttcggtg aaatttccga tccaagaaaa 660

ttgcatagag aaatcaaact gttagtgggc gtggtggaaa caggtttatt catcgacaac 720

gcttcaaaag cctacttcgg taattctgac ggtagtgttg aagttaccga aaagtga 777

<210> 19

<211> 370

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 19

Met Ser Glu Ser Pro Met Phe Ala Ala Asn Gly Met Pro Lys Val Asn

1 5 10 15

Gln Gly Ala Glu Glu Asp Val Arg Ile Leu Gly Tyr Asp Pro Leu Ala

20 25 30

Ser Pro Ala Leu Leu Gln Val Gln Ile Pro Ala Thr Pro Thr Ser Leu

35 40 45

Glu Thr Ala Lys Arg Gly Arg Arg Glu Ala Ile Asp Ile Ile Thr Gly

50 55 60

Lys Asp Asp Arg Val Leu Val Ile Val Gly Pro Cys Ser Ile His Asp

65 70 75 80

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

85 90 95

Glu Leu Lys Gly Asp Leu Ser Ile Ile Met Arg Ala Tyr Leu Glu Lys

100 105 110

Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro Asp Val

115 120 125

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

130 135 140

Phe Val Asn Leu Thr Asn Ile Gly Leu Pro Ile Gly Ser Glu Met Leu

145 150 155 160

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

165 170 175

Ile Gly Ala Arg Thr Thr Glu Ser Gln Leu His Arg Glu Leu Ala Ser

180 185 190

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

195 200 205

Asn Val Ala Val Asp Ala Cys Gln Ala Ala Ala His Ser His His Phe

210 215 220

Met Gly Val Thr Leu His Gly Val Ala Ala Ile Thr Thr Thr Lys Gly

225 230 235 240

Asn Glu His Cys Phe Val Ile Leu Arg Gly Gly Lys Lys Gly Thr Asn

245 250 255

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

260 265 270

Ser Asn Gly Leu Met Ile Asp Tyr Ser His Gly Asn Ser Asn Lys Asp

275 280 285

Phe Arg Asn Gln Pro Lys Val Asn Asp Val Val Cys Glu Gln Ile Ala

290 295 300

Asn Gly Glu Asn Ala Ile Thr Gly Val Met Ile Glu Ser Asn Ile Asn

305 310 315 320

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

325 330 335

Gly Val Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Thr Thr Glu Asp

340 345 350

Val Leu Arg Lys Leu Ala Ala Ala Val Arg Gln Arg Arg Glu Val Asn

355 360 365

Lys Lys

370

<210> 20

<211> 1113

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

atgagtgaat ctccaatgtt cgctgccaac ggcatgccaa aggtaaatca aggtgctgaa 60

gaagatgtca gaattttagg ttacgaccca ttagcttctc cagctctcct tcaagtgcaa 120

atcccagcca caccaacttc tttggaaact gccaagagag gtagaagaga agctatagat 180

attattaccg gtaaagacga cagagttctt gtcattgtcg gtccttgttc catccatgat 240

ctagaagccg ctcaagaata cgctttgaga ttaaagaaat tgtcagatga attaaaaggt 300

gatttatcca tcattatgag agcatacttg gagaagccaa gaacaaccgt cggctggaaa 360

ggtctaatta atgaccctga tgttaacaac actttcaaca tcaacaaggg tttgcaatcc 420

gctagacaat tgtttgtcaa cttgacaaat atcggtttgc caattggttc tgaaatgctt 480

gataccattt ctcctcaata cttggctgat ttggtctcct tcggtgccat tggtgccaga 540

accaccgaat ctcaactgca cagagaattg gcctccggtt tgtctttccc agttggtttc 600

aagaacggta ccgatggtac cttaaatgtt gctgtggatg cttgtcaagc cgctgctcat 660

tctcaccatt tcatgggtgt tactttgcat ggtgttgctg ctatcaccac tactaagggt 720

aacgaacact gcttcgttat tctaagaggt ggtaaaaagg gtaccaacta cgacgctaag 780

tccgttgcag aagctaaggc tcaattgcct gccggttcca acggtctaat gattgactac 840

tctcacggta actccaataa ggatttcaga aaccaaccaa aggtcaatga cgttgtttgt 900

gagcaaatcg ctaacggtga aaacgccatt accggtgtca tgattgaatc aaacatcaac 960

gaaggtaacc aaggcatccc agccgaaggt aaagccggct tgaaatatgg tgtttccatc 1020

actgatgctt gtataggttg ggaaactact gaagacgtct tgaggaaatt ggctgctgct 1080

gtcagacaaa gaagagaagt taacaagaaa tag 1113

<210> 21

<211> 256

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 21

Met Asp Phe Thr Lys Pro Glu Thr Val Leu Asn Leu Gln Asn Ile Arg

1 5 10 15

Asp Glu Leu Val Arg Met Glu Asp Ser Ile Ile Phe Lys Phe Ile Glu

20 25 30

Arg Ser His Phe Ala Thr Cys Pro Ser Val Tyr Glu Ala Asn His Pro

35 40 45

Gly Leu Glu Ile Pro Asn Phe Lys Gly Ser Phe Leu Asp Trp Ala Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ser Ile Asn Tyr Pro Gln Ile Leu Ala Pro Tyr Ala Pro Glu Val Asn

100 105 110

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

115 120 125

Ile Ser Lys Arg Asp Gly Asp Asp Lys Asn Asn Phe Ser Ser Val Ala

130 135 140

Thr Arg Asp Ile Glu Cys Leu Gln Ser Leu Ser Arg Arg Ile His Phe

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ala Glu Val Tyr Gly Val Asp Pro Thr Asn Glu Ser Gly Glu Arg Arg

210 215 220

Ile Thr Pro Glu Tyr Leu Val Lys Ile Tyr Lys Glu Ile Val Ile Pro

225 230 235 240

Ile Thr Lys Glu Val Glu Val Glu Tyr Leu Leu Arg Arg Leu Glu Glu

245 250 255

<210> 22

<211> 771

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 22

atggatttca caaaaccaga aactgtttta aatctacaaa atattagaga tgaattagtt 60

agaatggagg attcgatcat cttcaaattt attgagaggt cgcatttcgc cacatgtcct 120

tcagtttatg aggcaaacca tccaggttta gaaattccga attttaaagg atctttcttg 180

gattgggctc tttcaaatct tgaaattgcg cattctcgca tcagaagatt cgaatcacct 240

gatgaaactc ccttctttcc tgacaagatt cagaaatcat tcttaccgag cattaactac 300

ccacaaattt tggcgcctta tgccccagaa gttaattaca atgataaaat aaaaaaagtt 360

tatattgaaa agattatacc attaatttcg aaaagagatg gtgatgataa gaataacttc 420

agttctgttg ccactagaga tatagaatgt ttgcaaagct tgagtaggag aatccacttt 480

ggcaagtttg ttgctgaagc caagttccaa tcggatatcc cgctatacac aaagctgatc 540

aaaagtaaag atgtcgaggg gataatgaag aatatcacca attctgccgt tgaagaaaag 600

attctagaaa gattaactaa gaaggctgaa gtctatggtg tggaccctac caacgagtca 660

ggtgaaagaa ggattactcc agaatatttg gtaaaaattt ataaggaaat tgttatacct 720

atcactaagg aagttgaggt ggaatacttg ctaagaaggt tggaagagta a 771

<210> 23

<211> 370

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 23

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

1 5 10 15

Trp Arg Ile Lys Gly Tyr Asp Pro Leu Thr Pro Pro Asp Leu Leu Gln

20 25 30

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

35 40 45

Arg Asp Ser Val Cys Asp Ile Leu Asn Gly Lys Asp Asp Arg Leu Val

50 55 60

Ile Val Ile Gly Pro Cys Ser Leu His Asp Pro Lys Ala Ala Tyr Asp

65 70 75 80

Tyr Ala Asp Arg Leu Ala Lys Ile Ser Glu Lys Leu Ser Lys Asp Leu

85 90 95

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

100 105 110

Trp Lys Gly Leu Ile Asn Asp Pro Asp Met Asn Asn Ser Phe Gln Ile

115 120 125

Asn Lys Gly Leu Arg Ile Ser Arg Glu Met Phe Ile Arg Leu Val Glu

130 135 140

Lys Leu Pro Ile Ala Gly Glu Met Leu Asn Thr Ile Ser Pro Gln Phe

145 150 155 160

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

165 170 175

Ser Gln Leu His Arg Glu Leu Ala Ser Gly Leu Ser Phe Pro Ile Gly

180 185 190

Phe Lys Asn Gly Thr Asp Gly Gly Leu Gln Val Ala Ile Asp Ala Met

195 200 205

Arg Ala Ala Ala His Asp His Tyr Phe Leu Ser Val Thr Lys Pro Gly

210 215 220

Val Thr Ala Ile Val Gly Thr Glu Gly Asn Lys Asp Thr Phe Leu Ile

225 230 235 240

Leu Arg Gly Gly Lys Asn Gly Thr Asn Phe Asp Lys Glu Ser Val Gln

245 250 255

Asn Thr Lys Lys Gln Leu Glu Lys Ala Gly Leu Thr Asp Asp Ser Gln

260 265 270

Lys Arg Ile Met Ile Asp Cys Ser His Gly Asn Ser Asn Lys Asp Phe

275 280 285

Lys Asn Gln Pro Lys Val Ala Lys Cys Ile Tyr Asp Gln Leu Thr Glu

290 295 300

Gly Glu Asn Ser Leu Cys Gly Val Met Ile Glu Ser Asn Ile Asn Glu

305 310 315 320

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

325 330 335

Gly Cys Ser Val Thr Asp Ala Cys Ile Gly Trp Glu Thr Thr Glu Gln

340 345 350

Val Leu Glu Leu Leu Ala Glu Gly Val Arg Asn Arg Arg Lys Ala Leu

355 360 365

Lys Lys

370

<210> 24

<211> 1113

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 24

atgttcatta aaaacgatca cgccggtgac aggaaacgct tggaagactg gagaatcaaa 60

ggttatgatc cattaacccc tccagatctg cttcaacatg aatttccaat ttcagccaaa 120

ggtgaggaaa acattatcaa ggcaagagac tccgtctgtg atattttgaa tggtaaagat 180

gatcgtttag ttatcgtgat cgggccatgt tccctacatg accccaaagc cgcttacgat 240

tacgctgaca gattggctaa aatttcagaa aagttgtcaa aagacttatt gattattatg 300

agagcgtatt tagaaaaacc aaggactacc gttggctgga aagggttgat taacgaccct 360

gatatgaata actcttttca aatcaataaa ggtctacgga tttcgagaga aatgttcata 420

agactggttg aaaaattacc cattgctggt gaaatgctga acaccatttc tccgcagttt 480

ttgagtgatt gtttctcctt gggtgccatc ggtgctagaa ctactgaatc ccaactgcac 540

agagaattag catccggtct atctttccct attggattta agaacggtac tgatggtggt 600

ttgcaagtcg ccatcgacgc tatgagagcc gctgcacatg atcattactt cctttctgtc 660

acaaagccag gtgtcactgc tatcgtgggc actgaaggta acaaggatac cttcctgatc 720

ttgagaggtg gtaagaacgg tactaacttt gacaaagaaa gtgttcaaaa tactaagaaa 780

caattagaaa aggccggttt gactgacgat tcacagaaaa gaatcatgat cgattgttca 840

cacgggaaca gtaataaaga tttcaagaac caaccgaagg ttgccaaatg catttatgac 900

caactgacgg aaggtgaaaa tagtctttgt ggtgttatga ttgagtccaa cataaatgaa 960

ggtagacaag atattcctaa agaaggtggc agagagggat tgaagtatgg ttgttctgta 1020

acggatgctt gtattggttg ggagaccacc gaacaggtat tggagctatt ggccgaaggt 1080

gttagaaaca gaagaaaagc cttgaagaaa taa 1113

<210> 25

<211> 373

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 25

Met Val Ala Glu Leu Thr Ala Leu Arg Asp Gln Ile Asp Glu Val Asp

1 5 10 15

Lys Ala Leu Leu Asn Leu Leu Ala Lys Arg Leu Glu Leu Val Ala Glu

20 25 30

Val Gly Glu Val Lys Ser Arg Phe Gly Leu Pro Ile Tyr Val Pro Glu

35 40 45

Arg Glu Ala Ser Ile Leu Ala Ser Arg Arg Ala Gln Ala Glu Ala Leu

50 55 60

Gly Val Pro Pro Asp Leu Ile Glu Asp Val Leu Arg Arg Val Met Arg

65 70 75 80

Glu Ser Tyr Ser Ser Glu Asn Asp Lys Gly Phe Lys Thr Leu Cys Pro

85 90 95

Ser Leu Arg Pro Val Val Ile Val Gly Gly Gly Gly Gln Met Gly Arg

100 105 110

Leu Phe Glu Lys Met Leu Thr Leu Ser Gly Tyr Gln Val Arg Ile Leu

115 120 125

Glu Gln His Asp Trp Asp Arg Ala Ala Asp Ile Val Ala Asp Ala Gly

130 135 140

Met Val Ile Val Ser Val Pro Ile His Val Thr Glu Gln Val Ile Gly

145 150 155 160

Lys Leu Pro Pro Leu Pro Lys Asp Cys Ile Leu Val Asp Leu Ala Ser

165 170 175

Val Lys Asn Gly Pro Leu Gln Ala Met Leu Ala Ala His Asp Gly Pro

180 185 190

Val Leu Gly Leu His Pro Met Phe Gly Pro Asp Ser Gly Ser Leu Ala

195 200 205

Lys Gln Val Val Val Trp Cys Asp Gly Arg Lys Pro Glu Ala Tyr Gln

210 215 220

Trp Phe Leu Glu Gln Ile Gln Val Trp Gly Ala Arg Leu His Arg Ile

225 230 235 240

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

245 250 255

His Phe Ala Thr Phe Ala Tyr Gly Leu His Leu Ala Glu Glu Asn Val

260 265 270

Gln Leu Glu Gln Leu Leu Ala Leu Ser Ser Pro Ile Tyr Arg Leu Glu

275 280 285

Leu Ala Met Val Gly Arg Leu Phe Ala Gln Asp Pro Gln Leu Tyr Ala

290 295 300

Asp Ile Ile Met Ser Ser Glu Arg Asn Leu Ala Leu Ile Lys Arg Tyr

305 310 315 320

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

325 330 335

Gln Ala Phe Ile Asp Ser Phe Arg Lys Val Glu His Trp Phe Gly Asp

340 345 350

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

355 360 365

Asn Asp Asn Arg Gln

370

<210> 26

<211> 1122

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 26

atggttgctg aattgaccgc attacgcgat caaattgatg aagtcgataa agcgctgctg 60

aatttattag cgaagcgtct ggaactggtt gctgaagtgg gcgaggtgaa aagccgcttt 120

ggactgccta tttatgttcc ggagcgcgag gcatctatct tggcctcgag acgtgcacaa 180

gcggaagctc tgggtgtacc gccagatctg attgaggatg ttttgcgtcg ggtgatgcgt 240

gaatcttact ccagtgaaaa cgacaaagga tttaaaacac tttgtccgtc actgcgtccg 300

gtggttatcg tcggcggtgg cggtcagatg ggacgcctgt tcgagaagat gctgacacta 360

tcgggttatc aggtgcggat tctggagcaa catgactggg atcgagcggc tgatattgtt 420

gccgatgccg gaatggtgat tgttagtgtg ccaatccacg ttactgagca agttatcggc 480

aaattaccgc ctttaccgaa agattgtatt ctggttgatc tggcatcagt gaaaaatgga 540

ccattacagg ccatgctggc ggcgcacgat ggcccggtac tggggttaca cccgatgttc 600

ggcccggaca gcggtagcct ggcaaagcaa gttgtggtct ggtgtgatgg acgtaagccg 660

gaagcatacc aatggtttct ggagcaaatt caggtctggg gcgctcggct gcatcgtatt 720

agcgctgtcg agcacgatca gaatatggcg tttattcagg ctctgcgcca ctttgctact 780

tttgcttatg ggctgcatct ggcagaagaa aatgttcagc ttgagcaact tctggcgctc 840

tcttcgccga tttaccgcct tgagctggcg atggtcgggc gactgtttgc tcaggatccg 900

cagctttatg ccgacattat tatgtcgtca gagcgtaatc tggcgttaat caaacgttac 960

tataagcgtt tcggcgaggc gattgagttg ctggagcagg gcgataagca ggcgtttatt 1020

gacagtttcc gcaaggtgga gcactggttc ggcgattacg ttcagcgttt tcagagtgaa 1080

agccgcgtgt tattgcgtca ggcgaatgac aaccgccagt aa 1122

<210> 27

<211> 1378

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 27

accggagtgg ctctctttat caattaccgg gtttgataga atttgtccca tttttctttt 60

tctggttctc tatatgttat acaacaatgg aatagtcaaa atcctaggga tgttattaat 120

cggaaaacta ctatctcgaa cgaatagtta taattaatca atgaaaaaaa aaagagaaag 180

taaaaaatgt tacaacaact aatatacact aattgtcctt gataacagaa cttgttgtag 240

ttttattccg tggggaggag ggtgaatttt ttttatttag ttttaaagaa caaaacagaa 300

gtctatatac ttagcactat aatatccagt ttcaatttgc tctgtcagaa tccgaattgg 360

ctggttttct ttgttttacc acccccttcc atttatcagc atgatatttt ttttttagaa 420

taatcctttc aatcgttgaa gtagtttgtg ggaaaagaaa agttgttaaa ggcattaacg 480

tacacagtac tgaacggttg cattgataga ttttcattac ctctgaccac aatcctgagc 540

attggtatta tttgttttgc tattttcaga tcaaattact tgtaaaaaaa gaaatggtag 600

tatattggca ttgaacactg gctgttctat ttgtattact tttatatgta gacatatatt 660

attaggaatt tgtaatatca ctctaattat tatttgatat tctttctcta tctcccctgt 720

ttcttttgtc tatctctttc ttttttttta aacggccctt ttttaatggc attttctttt 780

tcttataatg cccacagttg aatgatttaa ctagcttttc actgacacat aaacaataat 840

gtatatgatc catcgaatga aatgacagtt ctattgcatt ttacctactt gtatattctg 900

gatactgcac aagaaaatga ccgcttccat caaaattcaa ccggatattg actctctaaa 960

gcaattacag cagcaaaatg acgatagttc cataaatatg tatcccgtgt atgcgtattt 1020

gccatcattg gatctgactc ctcacgtggc atatctaaaa ttggcacaat tgaacaaccc 1080

tgatagaaag gaatcatttc tgttggaaag tgctaagaca aataatgaat tagatcgtta 1140

ttcattcata ggtatctcgc cacgcaagac catcaaaacc ggtcctactg aaggcattga 1200

aacagatcct ttggaaattt tggaaaagga gatgtccacc tttaaagtag ccgaaaatgt 1260

tcctggttta ccgaagttaa gtggtggtgc tattggttat atttcttatg actgtgttcg 1320

ttatttcgag ccaaaaacaa gaaggccttt gaaagatgtc ctaagacttc cagaggca 1378

Claims

1.一种高产羟基酪醇的酿酒酵母，其特征在于，在能够合成酪醇的酿酒酵母基础上进一步表达如下任意组合的HpaB和HpaC：

(2)来源于大肠杆菌的HpaB和来源于铜绿假单胞菌的HpaC。

2.根据权利要求1所述高产羟基酪醇的酿酒酵母，其特征在于，所述来源于铜绿假单胞菌的HpaB为野生型或具有Q212D突变的突变型。

3.根据权利要求1所述高产羟基酪醇的酿酒酵母，其特征在于，所述能够合成酪醇的酿酒酵母经由如下任意一种方式改造获得：

(2)过表达酿酒酵母ARO2、ARO10、TKL1以及RKI1，过表达酿酒酵母ARO4^K229L、ARO7^G141S和ARO3^D154N突变体，以及敲除酿酒酵母PHA2和PDC1的表达；

4.根据权利要求1-3任意一项所述高产羟基酪醇的酿酒酵母，其特征在于，还包括：所述能够合成酪醇的酿酒酵母内源的TRP2基因的启动子为弱启动子，所述弱启动子为启动能力弱于原始启动子的启动子。

5.如下组合的HpaB和HpaC或它们的表达载体在构建高产羟基酪醇的酿酒酵母中的应用：

(2)来源于大肠杆菌的HpaB和来源于铜绿假单胞菌的HpaC。

6.权利要求1所述高产羟基酪醇的酿酒酵母的构建方法，其特征在于，将表达如下任意组合的HpaB和HpaC的重组载体转入能够合成酪醇的酿酒酵母中，获得所述高产羟基酪醇的酿酒酵母；

(2)来源于大肠杆菌的HpaB和来源于铜绿假单胞菌的HpaC。

7.根据权利要求6所述构建方法，其特征在于，所述能够合成酪醇的酿酒酵母经由如下任意一种方式改造获得：

8.根据权利要求6或7所述构建方法，其特征在于，还包括：将能够合成酪醇的酿酒酵母内源的TRP2基因的启动子更换为启动能力弱于原始启动子的启动子。

9.权利要求1-4任意一项所述高产羟基酪醇的酿酒酵母在制备羟基酪醇中的应用。

10.一种制备羟基酪醇的方法，其特征在于，采用权利要求1-4任意一项所述高产羟基酪醇的酿酒酵母进行发酵培养，收集发酵液上清分离出羟基酪醇。