CN107988128A - 一种产d-1,2,4-丁三醇的基因工程菌及其应用 - Google Patents
一种产d-1,2,4-丁三醇的基因工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种产D‑1,2,4‑丁三醇的基因工程菌及其应用,将2‑酮酸脱羧酶基因、木糖脱氢酶基因XylB、木糖酸脱水酶YjhG和醇脱氢酶基因YqhD转入宿主菌BL21(DE3)得到基因工程菌,所述2‑酮酸脱羧酶基因为来源于乳酸乳球菌(Lactococcus lactis)的α‑酮酸脱羧酶基因KdcA。经发酵培养生产D‑1,2,4‑丁三醇。本发明通过筛选所提供的α‑酮酸脱羧酶基因KdcA,能够有效提高D‑木糖合成D‑1,2,4‑丁三醇的能力,最终可达6.82g/L。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种产D-1,2,4-丁三醇的基因工程菌及其应用。
背景技术
D-1,2,4-丁三醇 (简称BT)是一种四碳的多元醇,性质与甘油类似,广泛应用于医药、农药、化妆品、造纸、高分子材料、烟草、军工等领域。BT及其相关衍生物是许多天然产物合成中的重要底物,也是许多手性化合物的合成前体:军事上,由于BT的硝基化合物冲击敏感性低、热稳定性好、低毒性、吸湿性好,与其它含能增塑剂混合使用,可显著提高以硝化纤维素为基火药的低温力学性能。因此,BT常作为 1,2,4-丁三醇三硝酸酯 (BTTN) 的合成前体,后者可代替***油作为推进剂和含能增塑剂,并具有更优的理化性质。医药方面,BT可被用作阳离子脂质体和多种药物的合成前体,BT还可以用来制备降胆固醇类药Movinolin、抗癌药物 compatin、治疗皮肤病药物羟基二十碳四烯酸(12-HETE)及艾滋病药物 3-羟基-四氢呋喃。此外,BT 还可用作可消除硝基化合物对人体的毒害,减少焦油成分危害的卷烟添加剂,可用作高分子材料的交联剂,增加材料的强度和硬度,还可用作高级墨水 的防干剂、高级服装表面处理剂、陶瓷加工助剂、特殊用途包装与储运等。BT能抑制微生物,是抗微生物制剂(如防腐剂)的组成部分。
2003 年,Niu等首先提出了BT的生物合成法。通过在大肠杆菌( Escherichia coli)中构建异源代谢途径,以 D-木糖或 L-***糖为底物,经过四步酶催化实现。整个工艺使用了莓实假单胞菌 Pseudomonas fragi 和大肠杆菌 Escherichia coli 两种微生物作为催化剂。之后,研究者将BT 转化途径的四步酶都在 E. coli 中进行表达,用 E. coli 即可完成整个 BT 的生物转化,大大简化了转化工艺。但从目前已有报道来看,用重组 E. coli 生产 BT 的催化效率较低,导致 BT产量无法满足工业生产的需求。如果要进一步提高 BT 的转化效率,首先必须对 BT 合成途径进行***地优化,找出催化限速步骤,精确调节催化各个步骤酶的表达量,以实现最大的转化通量。
BT是一种具有重要应用前景的多元醇,但作为一种非天然化合物,其生物转化途径仍面临着途径效率低的问题,因此对于 BT 合成途径的优化至关重要。
发明内容
针对现有BT生物转化途径仍面临的效率低的问题,对于 BT 合成途径中的2-酮酸脱羧酶进行筛选,以进一步提高 BT的产量。
本发明采用的技术方案为:
一种产D-1,2,4-丁三醇的基因工程菌,所述基因工程菌是将2-酮酸脱羧酶基因、木糖脱氢酶基因XylB、木糖酸脱水酶YjhG和醇脱氢酶基因YqhD转入宿主菌BL21(DE3)细胞内得到的基因工程菌。本发明考察了不同来源的2-酮酸脱羧酶基因对BT 合成产量的影响,例如:苯甲酰甲酸脱羧酶(恶臭假单胞菌,Pseudomonas putida), GenBank: AY143338.1、α-酮异戊酸脱羧酶(乳酸乳球菌KF147,Lactococcus lactis subsp. lactis (strain KF147), GenBank:ADA65057.1、α-酮酸脱羧酶(乳酸乳球菌,Lactococcus lactis),GenBank: AAS49166.1、苯丙酮酸脱羧酶(酿酒酵母,Saccharomyces cerevisiae),GenBank: KZV12623.1,得出所述2-酮酸脱羧酶基因采用来源于乳酸乳球菌的 α-酮酸脱羧酶时,BT产量最高,远远超过目前生物法BT的生产。
所述的宿主菌为大肠杆菌BL21(DE3)。
本发明中所述的木糖酸脱水酶YjhG Gene ID: 946829;所述的木糖脱氢酶XylB,Gene ID: 7329904;所述的醇脱氢酶YqhD,GenBank: ADK47404.1;所述的苯甲酰甲酸脱羧酶MdlC,GenBank: AY143338.1;所述的α-酮异戊酸脱羧酶KivD,GenBank: ADA65057.1;所述的α-酮酸脱羧酶KdcA,GenBank: AAS49166.1;所述的苯丙酮酸脱羧酶Aro10,GenBank:KZV12623.1。
来源于Lactococcus lactis的α-酮酸脱羧酶KdcA基因序列如SEQ.No.1所示;来源于Lactococcus lactis subsp. lactis (strain KF147)的α-酮异戊酸脱羧酶基因KivD序列如SEQ.No.2所示;来源于Pseudomonas putida的苯甲酰甲酸脱羧酶MdlC序列如SEQ.No.3所示;苯丙酮酸脱羧酶基因Aro10序列如SEQ.No.4所示
所述的产D-1,2,4-丁三醇的基因工程菌的构建方法,包括如下步骤:
1)构建克隆表达α-酮酸脱羧酶基因KdcA、木糖脱氢酶基因XylB、D-木糖酸脱水酶YjhG和醇脱氢酶基因YqhD;
2)KdcA基因被***质粒pTRC99a 的NcoⅠ和Sac Ⅰ位点之间,得到质粒pTRC-KdcA,XylB基因被***质粒pTRC99a 的NcoⅠ和BamHⅠ位点之间,从而获得pTRC99a-XylB。然后通过PCR得到带有TRC启动子的XylB片段,将该片段***质粒pTRC-KdcA的Sac Ⅰ和BamH Ⅰ位点之间,得到质粒pTRC-KdcA-TRC-XylB。
3)将基因片段YjhG、YqhD分别***pCWJ质粒,到重组质粒 pCWJ-YjhG和pCWJ-YqhD;以pCWJ-YqhD质粒为模板扩增出带有TRC启动子的YqhD片段,将片段***质粒pCWJ-YjhG,得到质粒pCWJ-YjhG-TRC-YqhD。
4)将质粒pTRC-KdcA-TRC-XylB和质粒pCWJ-YjhG-TRC-YqhD共同转入宿主菌BL21(DE3)细胞内得产D-1,2,4-丁三醇的基因工程菌。
所述的基因工程菌在发酵生产D-1,2,4-丁三醇中的应用。将基因工程菌接种至发酵培养基中,加入IPTG诱导发酵得产物D-1,2,4-丁三醇。
所述发酵培养基为: 蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L, D-木糖 25g/L,氨苄霉素100 mg/L, 氯霉素 68 mg/L 。
所述发酵步骤控制发酵温度为30-37℃,发酵时间为60-72h。
所述IPTG诱导浓度为0.1~1.25mmol/L,优选为1mmol/L。
本发明要求保护一种来源于乳酸乳球菌(Lactococcus lactis)的α-酮酸脱羧酶在D-1,2,4-丁三醇生产过程中的应用。分别构建克隆表达不同的2-酮酸脱羧酶基因(MdlC、KivD、 KdcA和Aro10),并将构建好的基因转入宿主菌BL21(DE3)细胞内得基因工程菌,培养基因工程菌并接种至发酵培养基中生产BT。
本发明首先识别整个催化过程的限制性步骤,Cao等利用重组 E. coli 转化木糖生产木糖酸可达到理论得率的近 88%,Valdehuesa 等报道了用重组 E. coli 转化木糖生产 BT 时,有 82.28%的木糖转化为木糖酸,但只有 10.25%的木糖最终转化为 BT,这表明后三步酶催化可能是整个途径的限速步骤。由于第三步脱羧反应是非天然催化反应,同时脱羧反应又是不可逆反应,可推测该步反应极有可能是整个反应的限制性步骤,因此,本发明考察了苯甲酰甲酸脱羧酶(Pseudomonas putida), GenBank: AY143338.1、α-酮异戊酸脱羧酶(Lactococcus lactis subsp. lactis (strain KF147), GenBank: ADA65057.1、α-酮酸脱羧酶(Lactococcus lactis), GenBank: AAS49166.1、苯丙酮酸脱羧酶(Saccharomyces cerevisiae), GenBank: KZV12623.1;对合成途径进行新一轮的优化,以进一步提高 BT 的产量。
有益效果
本发明通过筛选不同的2-酮酸脱羧酶,优化了D-木糖生产D-1,2,4-丁三醇的路径,采用来源于乳酸乳球菌的 α-酮酸脱羧酶构建基因工程菌,D-1,2,4-丁三醇的产量显著提高,最终可达6.82g/L。
附图说明
图1为乳酸乳球菌KdcA对发酵生产BT的影响;
图2为不同的2-酮酸脱羧酶对发酵生产BT的影响;
图3为IPTG浓度对发酵生产BT的影响。
具体实施方式
下面的实施例可使本专业技术人员更全面地理解本发明,但不以任何方式限制本发明。本发明中,重组表达载体可以用本领域熟知的方法导入宿主细胞中,这些方法包括:氯化钙热激法,电转化法,PEG介导法,基因枪法等。
本发明的实施将采用本领域技术人员的能力范围之内的化学、分子生物学等领域的传统技术。另外,除非另有说明,在本文中,核酸以5’至3’的方向从左向右书写,氨基酸序列则以氨基端到羧基端的方向从左向右书写。
下面结合实验室具体的实验数据进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件操作,例如《分子克隆实验指南》,或者制造厂商所建议的条件。
实施例1 构建基因工程菌BL21-02、 BL21-08、BL21-09、 BL21-10
1)构建克隆表达2-酮酸脱羧酶基因MdlC、KivD、KdcA、Aro10;木糖脱氢酶基因XylB、D-木糖酸脱水酶YjhG和醇脱氢酶基因YqhD;D-木糖酸脱水酶YjhG (Escherichia coli),GeneID: 946829;所述的木糖脱氢酶基因XylB,Gene ID: 7329904;所述的醇脱氢酶基因YqhD,GenBank: ADK47404.1;所述α-酮酸脱羧酶 (Lactococcus lactis), GenBank:AAS49166.1;
2) 2-酮酸脱羧酶基因MdlC、KivD、KdcA、Aro10分别被***质粒pTRC99a (TransGen)的NcoⅠ和Sac Ⅰ位点之间,得到质粒pTRC-MdlC、pTRC-KivD、pTRC-KdcA、pTRC-Aro10,XylB基因被***质粒pTRC99a 的NcoⅠ和BamHⅠ位点之间,从而获得pTRC99a-XylB。然后通过PCR得到带有TRC启动子的XylB片段,将该片段分别***质粒pTRC-MdlC、pTRC-KivD、pTRC-KdcA、pTRC-Aro10的Sac Ⅰ和BamH Ⅰ位点之间,得到质粒pTRC-MdlC-TRC-XylB、pTRC-KivD-TRC-XylB、pTRC-KdcA-TRC-XylB、pTRC-Aro10-TRC-XylB。
3)将基因片段YjhG、YqhD分别***pCWJ质粒,得到重组质粒 pCWJ-YjhG和pCWJ-YqhD。以pCWJ-YqhD质粒为模板扩增出带有TRC启动子的YqhD片段,将片段***质粒pCWJ-YjhG,得到质粒pCWJ-YjhG-TRC-YqhD。
4)将质粒pTRC-MdlC-TRC-XylB、pTRC-KivD-TRC-XylB、pTRC-KdcA-TRC-XylB、pTRC-Aro10-TRC-XylB分别和质粒pCWJ-YjhG-TRC-YqhD共同转入宿主菌BL21(DE3)细胞内得基因工程菌BL21-02、 BL21-08、BL21-09、 BL21-10。
所述构建方法中采用不同来源的2-酮酸脱羧酶基因,构建得到不同的基因工程菌,其中,2-酮酸脱羧酶分别是苯甲酰甲酸脱羧酶(Pseudomonas putida) MdlC, GenBank:AY143338.1、α-酮异戊酸脱羧酶(Lactococcus lactis subsp. lactis (strain KF147))KivD, GenBank: AIS03677.1、α-酮酸脱羧酶(Lactococcus lactis), GenBank:AJG88423.1、苯丙酮酸脱羧酶(Saccharomyces cerevisiae),GenBank: KZV12623.1。
实施例2 基因工程菌发酵生产D-1,2,4-丁三醇
平板培养:
将储存于-80℃的甘油冻存菌取出,在平板培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl10 g/L,氨苄霉素100 mg/L, 氯霉素 68 mg/L )上进行划线操作,然后将平板置于37℃培养箱培养12-16h。
种子液培养:
向50 mL离心管中加入5 mL种子培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L,氨苄霉素100 mg/L, 氯霉素 68 mg/L ),挑取少量甘油冻存菌进行接种,在200 rpm、37 ℃条件下培养10~12 h。
摇瓶发酵培养:
在500 mL摇瓶中装入50 mL发酵培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L,D-木糖 25g/L ,氨苄霉素100 mg/L, 氯霉素 68 mg/L),接种量为1 %,转速为200 rpm,培养温度为37℃,发酵初始时加入终浓度为1mmol/L的IPTG诱导,并加入10g/LCaCO3作为缓冲,发酵60~72h。每隔12h取样1-2mL,离心取上清,进行液相检测。
通过发酵结果比较(图1、图2),来源于Lactococcus lactis的α-酮酸脱羧酶KdcA的基因工程菌有更高的催化效率,能够提高重组菌株合成BT的能力,D-1,2,4-丁三醇产量达到6.82g/L,摩尔收率为38.59%。比来源于Pseudomonas putida的苯甲酰甲酸脱羧酶MdlC和来源于Lactococcus lactis subsp. lactis (strain KF147)的α-酮异戊酸脱羧酶KivD分别提高了102%,58.97%。
实施例3 IPTG浓度对基因工程菌BL21-09发酵产BT的影响
IPTG为异丙基硫代-β-D-半乳糖,是一种十分有效的乳糖操纵子的诱导剂,可与阻遏物结合促进基因的表达。以IPTG作为诱导剂,其作用是与阻遏物结合,起解阻遏作用。因此当所加入的IPTG量足以封闭所有阻遏物位点时,即为理论上的最佳浓度。但是IPTG浓度过高时,将会作为毒性物质,影响细胞的繁殖和生长。本发明以BL21-09作为实验菌株,考察了不同浓度的IPTG对BT合成的影响,设置的浓度范围为0.1~1.25mmol/L,结果如图3所示。当诱导剂浓度低于1mmol/L时,BT产量是随着IPTG的浓度增加而增加的。浓度达到1mmol/L时,BT的产量最高,浓度超过1mmol/L达到1.25mmol/L时,产物产量降低,说明此时诱导剂对菌株产生了一些抑制作用。
D-1,2,4-丁三醇的高效液相色谱检测方法:
D-1,2,4-丁三醇的检测条件为:Agilent Technologies1290 高效液相色谱;Bio-RadAminex® HPX-87H IonExclusion Column (300 mm × 7.8 mm) 有机酸柱;流动相为5mmol/L H2SO4;流速0.6 mL/min,柱温55 ℃,进样量20 μL,
示差检测器。
实施例4 基因工程菌BL21-09发酵生产D-1,2,4-丁三醇
将储存于-80℃的甘油冻存菌取出,在平板培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl10 g/L,氨苄霉素100 mg/L, 氯霉素 68 mg/L )上进行划线操作,然后将平板置于37℃培养箱培养12-16h。
向50 mL离心管中加入5 mL种子培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L,氨苄霉素100 mg/L, 氯霉素 68 mg/L ),挑取少量甘油冻存菌进行接种,在200 rpm、37℃条件下培养10~12 h。
在500 mL摇瓶中装入50 mL发酵培养基(蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L, D-木糖 25g/L ,氨苄霉素100 mg/L, 氯霉素 68 mg/L),接种量为1 %,转速为200 rpm,培养温度为37℃,发酵初始时加入终浓度为1mmol/L的IPTG诱导,并加入10g/LCaCO3作为缓冲,发酵60h,D-1,2,4-丁三醇的产量达6.82g/L,摩尔收率38.59%。
序列表
<110> 南京工业大学
<120> 一种产D-1,2,4-丁三醇的基因工程菌及其应用
<141> 2017-11-27
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1644
<212> DNA
<213> 乳酸乳球菌(Lactococcus lactis)
<400> 1
atgtataccg tgggcgatta tctgctggat cgcctgcatg aactgggcat cgaagaaatt 60
tttggcgtgc cgggcgatta taatctgcag ttcctggatc agattattag ccgcgaagac 120
atgaaatgga ttggcaatgc gaatgaactg aatgcgagct atatggcgga tggctatgcg 180
cgcaccaaaa aagcagcggc gtttctgact acctttggcg tgggtgaact gagcgcgatt 240
aatggtctgg cgggcagcta tgcagaaaat ctgccggtgg tggaaattgt tggtagcccg 300
accagcaaag tgcagaatga tggcaaattt gtgcatcata ccctggcgga tggcgatttt 360
aaacacttca tgaagatgca tgaaccggtt accgcggcgc gtactttatt gaccgcggaa 420
aatgcgacct atgaaattga tcgcgtgctg agccagttgc tgaaagaacg caaaccggtg 480
tatattaatc tgccggtgga tgttgcggca gcgaaagcgg aaaaaccagc gctgagcctg 540
gaaaaagaaa gcagcaccac caataccacc gaacaggtga ttctgagcaa aattgaggag 600
agcctgaaaa atgcgcagaa accggtggtt attgcgggcc atgaagtgat tagctttggc 660
ctggaaaaaa ccgtgaccca gtttgtgagc gaaaccaaac tgccgattac caccctgaat 720
tttggcaaaa gcgcggtgga tgaaagcctg ccgagctttc tgggcattta taatggcaag 780
ctgagcgaaa ttagcctgaa gaactttgtg gaaagcgcgg attttattct gatgctgggc 840
gtgaaactga ccgatagcag caccggcgcg tttacccatc atctggacga gaataagatg 900
atcagcctga atattgacga gggcatcatc tttaacaagg tggtggagga ttttgatttt 960
cgcgcggtgg ttagcagtct gagcgaactg aaaggcattg aatatgaggg ccagtatatc 1020
gataaacagt acgaggagtt tattccgagc agcgcgccat taagccagga tcgtttgtgg 1080
caagcggtgg aaagcctgac ccagagcaat gaaaccattg tggcggaaca gggcaccagt 1140
ttttttggcg cgagcaccat ttttctgaaa agcaacagcc gctttattgg ccaaccactg 1200
tggggcagca ttggctatac ctttccagcg gcgttgggta gccagattgc ggataaagaa 1260
agccgccatc tgctgtttat tggcgatggc agcttgcaac tgaccgtgca agaactgggc 1320
ctgagcattc gcgaaaaact gaatccgatc tgcttcatca tcaacaacga tggctatacc 1380
gtggaacgcg aaattcatgg cccgacccag agctataatg atatcccgat gtggaattat 1440
agcaaactgc cggaaacctt tggcgcgacc gaagatcgtg tggtgagcaa aattgtgcgc 1500
accgagaatg aatttgtgag cgtgatgaaa gaagcgcagg cggatgtgaa tcgcatgtat 1560
tggattgaac tggtgctgga aaaagaagat gcgccgaaac tgctgaaaaa aatgggcaaa 1620
ctgtttgcgg aacagaacaa gtga 1644
<210> 2
<211> 1647
<212> DNA
<213> 乳酸乳球菌(Lactococcus lactis)
<400> 2
atgtataccg tgggcgatta tctgctggat cgcctgcatg aactgggcat cgaagaaatt 60
tttggcgtgc cgggcgatta taatctgcag ttcctggatc agattattag ccgcaaggat 120
atgaaatggg tgggcaatgc gaatgaactg aatgcgagct atatggcgga tggctatgcg 180
cgcaccaaaa aagcggcggc gtttctgact acctttggcg ttggtgaact gagcgcggtt 240
aatggtctgg cgggcagcta tgcagaaaat ctgccggtgg tggaaattgt tggtagcccg 300
accagcaaag tgcagaatga aggcaaattt gtgcatcata ccctggcgga tggcgatttt 360
aagcacttca tgaagatgca tgaaccggtt accgcggcgc gtaccttatt gaccgcggaa 420
aatgcgaccg tggaaattga tcgcgtgctg agcgcgttac tgaaagaacg caagccggtg 480
tatattaatc tgccggtgga tgttgcggca gcgaaagcgg aaaaaccaag cctgccgctg 540
aaaaaagaaa atccgaccag caataccagc gatcaggaaa tcctgaataa aatccaggag 600
agcctgaaaa atgcgaaaaa gccgattgtg attaccggcc acgaaattat tagcttcggc 660
ctggaaaata ccgtgaccca gtttattagc aaaaccaagc tgccgattac caccctgaat 720
tttggcaaaa gcagcgtgga tgaaaccctg ccgagctttc tgggcattta taatggcaaa 780
ctgagcgaac cgaacctgaa agaatttgtg gaaagcgcgg attttattct gatgctgggc 840
gtgaaactga ccgatagcag cactggcgcg tttacccatc atctgaacga gaacaagatg 900
atcagcctga atatcgacga aggcaagatt tttaacgaaa gcatccagaa ctttgacttt 960
gaaagcctga ttagcagcct gctggatctg agcggcattg aatataaggg caagtacatc 1020
gataagaagc aggaggattt tgtgccgagc aatgcgctgt taagccagga tcgtctgtgg 1080
caggcggttg aaaatctgac ccagagcaat gaaaccattg tggcggaaca gggcaccagc 1140
ttttttggcg cgagcagcat ttttctgaaa ccgaagagcc attttattgg ccagccgctg 1200
tggggtagca ttggctatac ctttccagca gcgctgggca gtcaaattgc ggataaagaa 1260
agccgccatc tgctgtttat tggcgatggc agcctgcaac tgaccgtgca agaactgggt 1320
ctggcgattc gcgaaaaaat taacccgatc tgcttcatca tcaacaatga tggctatacc 1380
gtggaacgcg aaattcatgg cccgaatcag agctataatg atatcccgat gtggaattat 1440
agcaaactgc cggaaagctt tggcgcgacc gaagaacgtg tggtgagcaa aattgtgcgc 1500
accgagaatg aatttgtgag cgtgatgaaa gaagcgcagg ccgatccgaa tcgcatgtat 1560
tggattgaac tggtgctggc gaaagaagat gcgccgaaag tgctgaaaaa aatgggcaaa 1620
ctgtttgcgg aacagaacaa gagctaa 1647
<210> 3
<211> 1587
<212> DNA
<213> 恶臭假单胞菌(Pseudomonas putida)
<400> 3
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgcttggaa ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctctgctg accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtgct 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 4
<211> 1908
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 4
atggcaccgg tgaccattga aaaattcgtg aaccaggaag aacgccatct ggtgagcaat 60
cgcagcgcga ccattccatt tggcgagtac atttttaaac gcctgctgag cattgatacc 120
aaaagcgtgt ttggcgtgcc gggcgatttt aatctgagcc tgctggaata tctgtatagc 180
ccgagcgttg aaagcgcggg cttacgttgg gttggcacct gcaatgaact gaatgcggcg 240
tatgcggcgg atggttatag ccgctacagc aataaaattg gctgcctgat taccacctat 300
ggcgtgggtg aactgagtgc gctgaatggt attgcgggca gctttgcgga aaatgtgaag 360
gtgctgcata ttgtgggcgt ggcgaaaagc attgatagcc gcagcagcaa ttttagcgat 420
cgcaatctgc atcatctggt gccgcagctg catgatagca attttaaagg cccgaaccat 480
aaagtgtatc acgacatggt gaaagatcgc gtggcatgca gtgtggcgta tctggaagat 540
attgaaaccg cgtgcgatca ggtggataat gtgatccgcg acatttacaa gtatagcaag 600
ccgggctata tttttgtgcc ggcggatttt gcggatatga gcgtgacctg cgataatctg 660
gtgaatgtgc cgcgcattag ccagcaggat tgcattgtgt atccgagcga aaatcagctg 720
agcgacatca ttaataagat caccagctgg atctatagca gcaaaacccc ggcgattctg 780
ggtgatgtgc tgaccgatcg ctatggcgtg agcaattttc tgaacaagct gatttgcaaa 840
accggcatct ggaattttag caccgtgatg ggcaaaagcg tgattgatga aagcaatccg 900
acctatatgg gccagtataa tggcaaagaa ggcctgaaac aggtgtatga gcattttgaa 960
ctgtgcgatc tggtgctgca ttttggcgtg gatatcaacg aaatcaacaa cggccattac 1020
accttcacct ataaaccgaa cgcgaagatt attcagttcc acccgaatta tattcgcctg 1080
gtggataccc gtcagggcaa tgaacagatg ttcaagggca ttaattttgc gccgatcctg 1140
aaagaactgt acaagcgcat tgatgtgagc aaactgagcc tgcagtatga tagcaatgtg 1200
acccagtata ccaatgaaac catgcgcctg gaagatccga ccaatggcca gagcagcatt 1260
attacccagg tgcatctgca gaaaaccatg ccgaaatttc tgaatccggg cgatgtggtg 1320
gtttgtgaaa ccggcagctt tcagtttagc gtgcgcgatt ttgcgtttcc gagccagctg 1380
aaatatatta gccagggctt ctttctgagc attggcatgg cattgccagc agcgttaggt 1440
gttggcattg cgatgcagga tcatagcaac gcgcatatta atggcggcaa cgtgaaggaa 1500
gattataaac cgcgcctgat tctgtttgaa ggcgatggtg cggcgcaaat gaccattcag 1560
gaactgagca ccattctgaa atgcaatatt ccgctggaag tgatcatttg gaacaacaac 1620
ggctatacca ttgaacgcgc gattatgggt ccaacccgca gctataatga tgtgatgagc 1680
tggaaatgga ccaaactgtt tgaagcgttc ggcgattttg atggcaaata caccaatagc 1740
accctgattc agtgcccgag caaactggcg ctgaaactgg aagaactgaa aaacagcaat 1800
aaacgcagcg gcattgaact gctggaagtg aaactgggcg aactggattt tccggaacag 1860
ctgaaatgca tggttgaagc ggcagcgctg aaacgcaata agaagtga 1908
Claims (8)
1.一种产D-1,2,4-丁三醇的基因工程菌,所述基因工程菌是将2-酮酸脱羧酶基因、木糖脱氢酶基因XylB、木糖酸脱水酶YjhG和醇脱氢酶基因YqhD转入宿主菌BL21(DE3)得到基因工程菌,其特征在于,所述2-酮酸脱羧酶基因为来源于乳酸乳球菌(Lactococcus lactis)的α-酮酸脱羧酶基因KdcA。
2.根据权利要求1所述的一种产D-1,2,4-丁三醇的基因工程菌,其特征在于,所述α-酮酸脱羧酶基因KdcA序列如SEQ.No.1所示。
3.根据权利要求1所述的一种产D-1,2,4-丁三醇的基因工程菌,其特征在于,所述宿主菌为大肠杆菌BL21(DE3)。
4.权利要求1或2所述的产D-1,2,4-丁三醇的基因工程菌的构建方法,其特征在于,包括如下步骤:
1)构建克隆表达α-酮酸脱羧酶基因KdcA、木糖脱氢酶基因XylB、D-木糖酸脱水酶YjhG和醇脱氢酶基因YqhD;
2)KdcA基因被***质粒pTRC99a 的NcoⅠ和Sac Ⅰ位点之间,得到质粒pTRC-KdcA,XylB基因被***质粒pTRC99a 的NcoⅠ和BamHⅠ位点之间,从而获得pTRC99a-XylB;然后通过PCR得到带有TRC启动子的XylB片段,将该片段***质粒pTRC-KdcA的Sac Ⅰ和BamH Ⅰ位点之间,得到质粒pTRC-KdcA-TRC-XylB;
3)将基因片段YjhG、YqhD分别***pCWJ质粒, 到重组质粒 pCWJ-YjhG和pCWJ-YqhD;以pCWJ-YqhD质粒为模板扩增出带有TRC启动子的YqhD片段,将片段***质粒pCWJ-YjhG,得到质粒pCWJ-YjhG-TRC-YqhD;
4)将质粒pTRC-KdcA-TRC-XylB和质粒pCWJ-YjhG-TRC-YqhD共同转入宿主菌BL21(DE3)细胞内得基因工程菌BL21-09。
5.权利要求1所述的基因工程菌在发酵生产D-1,2,4-丁三醇中的应用。
6.根据权利要求5所述的应用,其特征在于,将基因工程菌接种至发酵培养基中,加入IPTG诱导发酵得产物D-1,2,4-丁三醇。
7.根据权利要求6所述的应用,其特征在于,所述发酵培养基为: 蛋白胨 10g/L,酵母粉 5g/L,NaCl 10 g/L, D-木糖 25g/L ,氨苄霉素100 mg/L, 氯霉素 68 mg/L 。
8.根据权利要求6所述的应用,其特征在于,发酵温度为30-37℃,发酵时间为60-72h。
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