CN105802986A - 一种产靛蓝色素基因工程菌的构建方法与应用 - Google Patents

一种产靛蓝色素基因工程菌的构建方法与应用 Download PDF

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CN105802986A
CN105802986A CN201610219467.4A CN201610219467A CN105802986A CN 105802986 A CN105802986 A CN 105802986A CN 201610219467 A CN201610219467 A CN 201610219467A CN 105802986 A CN105802986 A CN 105802986A
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styab
pseudomonas putida
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indigo
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王成涛
尹胜
郝帅
程雷
孙宝国
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Abstract

本发明涉及一种重组Pseudomonas putida基因工程菌株及其制备方法，该工程菌株携带超量表达内源苯乙烯单加氧酶基因styAB的表达载体。该工程菌株的制备方法包括如下步骤：1)利用PCR方法从Pseudomonas putida基因组中扩增内源苯乙烯单加氧酶基因styAB片段；2)设计特定的限制性酶切位点，构建到Pseudomonas质粒载体pBBR1MCS‑2上；3)通过电击转化方法将重组质粒转化到Pseudomonas putida中。通过本发明制备的重组Pseudomonas putida工程菌株能显著提高天然色素靛蓝的合成量，能应用于食品、医药和日化等工业中。

Description

一种产靛蓝色素基因工程菌的构建方法与应用
技术领域
本发明涉及一种产靛蓝色素基因工程菌的构建方法及其应用，属于食品添加剂制造技术领域，制备的靛蓝色素与植物靛蓝的化学结构相同，为天然食用色素，该色素产品可应用于食品、保健品、日化和医药等行业。
背景技术
食用色素是一类重要食品添加剂，广泛应用于食品、医药、化妆品、印染等工业领域。根据颜色技术原理，红、黄、蓝为基本三原色，三原色根据不同浓度组合可调配出除白色外的各种不同色调。全球对染料的年需求是80万吨，其中蓝色素超过8万吨，目前主要通过化学合成制造。化学合成蓝色素需要使用苯胺、硝基苯、邻苯二甲酸酐等原料，毒性大、污染环境严重。我国使用的食品蓝色素主要是“亮蓝”(GB7655.1-2005)和“亮蓝铝色淀”(GB7655.2-2005)，美国称为靛蓝素(Indigotine；FD&CBlueNo.2，USA)，均属于化学合成色素。目前天然食用蓝色素来源极少，主要是从木蓝、菘蓝、蓼蓝、马蓝等植物中提取的靛蓝色素，靛蓝颜色鲜艳而耐久，作为染料使用已有2500年的历史。随着生活水平的提高和人们对健康的日益关注，“天然”、“绿色”已成为社会的崇尚和普遍追求，食品、化妆品等生产更倾向于使用天然色素，但由于受资源、成本等限制，天然色素价格昂贵。因此天然色素及其绿色生产技术的开发和应用已成为时代的要求。微生物合成天然色素不受原料资源限制，符合“环境友好型”要求，是近年来天然色素制造的重要发展方向。
目前已发现多种微生物(如Pseudomonassp.，Acinetobactersp.，Bacillusmegatreium等)具有生物合成靛蓝色素的能力。微生物中靛蓝的生物合成主要是通过不同的加氧酶(萘双加氧酶、苯酚羟化酶、二甲苯单加氧酶、黄素蛋白单加氧酶、细胞色素P450单加氧酶、铜依赖单加氧酶等)以吲哚为底物通过加氧(羟化)反应来实现。但由于野生型菌株中加氧酶的活力较低，导致生物转化靛蓝色素产量较低。文献报道在Pseudomonassp.中靛蓝色素的生物合成主要有两种途径。一种途径是在单加氧酶和异构酶的催化作用下，吲哚被氧化为羟基吲哚，羟基吲哚聚合成靛蓝分子；另外一种途径是在双加氧酶和异构酶的催化作用下，吲哚被氧化为羟基吲哚和2-羟基吲哚，羟基吲哚聚合成靛蓝分子，羟基吲哚和2-羟基吲哚聚合成靛玉红分子，终产物为靛蓝和靛玉红的混合物，难以分离纯化。因此，利用只具有单加氧酶催化活性的Pseudomonas菌株以吲哚为底物进行发酵能够获得单一的天然靛蓝色素分子。
本课题组分离筛选出一株具有较高单加氧酶活性的PseudomonasputidaB4，具有一定的靛蓝生物合成能力。课题组研究发现，该单加氧酶为苯乙烯单加氧酶，由styAB基因编码，并且是P.putidaB4合成天然靛蓝色素的关键限速酶。因此，构建单加氧酶编码基因超量表达的基因工程菌株，大量提高单加氧酶的催化活性，可显著促进靛蓝色素生物合成代谢流量的增强和产量的提高。
本申请专利通过在P.putidaB4中对靛蓝色素合成关键限速酶苯乙烯单加氧酶编码基因styAB进行超量表达，增强目的产物靛蓝色素的代谢流量，提高其产量，构建高产靛蓝色素的工程菌株，并利用上述工程菌株通过吲哚发酵生产天然的靛蓝色素。该发明为代谢工程菌及细胞炼制工厂的定向设计构建、天然靛蓝色素系生产提供理论基础和技术指导。
发明内容
本发明提供一种高产天然靛蓝色素的Pseudomonasputida基因工程菌的构建方法与应用。以PseudomonasputidaB4为出发菌株，克隆了靛蓝色素合成代谢途径中的关键限速酶苯乙烯单加氧酶编码基因styAB，通过增强关键酶基因的表达，显著提高了工程菌株B4-01中靛蓝色素合成量，能够在以吲哚为底物的发酵体系中高效生产天然的靛蓝色素。
生物材料保藏相关情况的说明
生物材料保藏单位：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位地址：北京市朝阳区北辰西路1号院3号
保藏日期：2016年03月10日
保藏编号：CGMCCNo.12202
分类命名：PseudomonasputidaB4-01
附图说明
图1重组质粒载体的构建
pBBR1MCS-2：E.coli-Pseudomonas穿梭质粒载体
pBBR1MCS-2-styAB：携带styAB基因的重组质粒载体
图2Pseudomonasputida菌株中单加氧酶的活性检测
野生型菌株B4
■：转入空质粒载体pBBR1MCS-2的重组菌株B4-0
转入重组质粒载体pBBR1MCS-2-styAB的重组菌株B4-01
图3Pseudomonasputida菌株中靛蓝的合成
◇：野生型菌株B4
△：转入空质粒载体pBBR1MCS-2的重组菌株B4-0
■：转入重组质粒载体pBBR1MCS-2-styAB的重组菌株B4-01
具体实施方式
1.styAB基因的克隆
1.1PseudomonasputidaB4基因组DNA的提取
将新鲜活化的PseudomonasputidaB4菌种接种至LB液体培养基中，置于30℃摇床中，220rpm震荡培养过夜。
(1)取1mL菌液至1.5mL离心管中，常温12000rpm离心1min，弃上清；
(2)采用细菌基因组DNA提取试剂盒(天根生化科技(北京)有限公司)提取基因组DNA。
1.2styAB基因的克隆
设计特异性引物，以PseudomonasputidaB4基因组DNA为模板，利用PCR方法扩增styAB基因片段。
styAB基因引物序列：
上游引物：5’-CGGGGTACCAGCAACCCACAACAAAAACAAG-3’
下游引物：5’-CCCAAGCTTGTGATCGGCACAGAAAGG-3’
PCR反应体系
引物序列中引入了KpnI和HindIII酶切位点，PCR程序如下：
95℃预变性5min，94℃变性30s，57℃退火30s，72℃延伸1min30s，共30个循环，72℃终延伸10min。
扩增产物经过双酶切形成粘性末端，酶切体系如下：两种限制性内切酶各2μL，10×Buffer5μL，质粒DNA10μL，无菌水31μL，37℃水浴2h。
2.styAB基因超量表达重组质粒载体的构建
使用KpnI和HindIII对质粒pBBR1MCS-2进行酶切(酶切体系同1.2)，形成粘性末端，与经过酶切的styAB基因片段连接，构建重组质粒pBBR1MCS-2-styAB；具体见图1。
连接体系：载体DNA1μL，目的基因片段7μL，10×T4ligaseBuffer1μL，T4DNAligase1μL，16℃空气浴连接过夜。
3.基因工程菌株的构建
利用经典的电击转化方法分别将质粒载体pBBR1MCS-2和pBBR1MCS-2-styAB转入野生型菌株B4中，分别构建重组菌株B4-0和B4-01。
电击参数：电压1.5kV，电阻400Ω，电容25μF.
Pseudomonasputida感受态细胞的制备：
(1)挑取单菌落接种于LB培养基中，置于30℃摇床中，220rpm震荡培养过夜；
(2)取2mL过夜培养物转接于200mLLB培养基中，在37℃摇床上剧烈振荡培养至OD600＝0.6；
(3)将菌液迅速置于冰上，在超净工作台和冰上操作以下步骤；
(4)吸取1.5mL培养好的菌液至1.5mL离心管中，在冰上冷却10min；
(5)4℃下3000g冷冻离心5min；
(6)弃去上清，加入1500μL冰冷的10％甘油，用移液枪轻轻上下吸动打匀，使细胞重新悬浮；
(7)4℃下3000g冷冻离心5min；
(8)弃去上清，加入750μL冰冷的10％甘油，用移液枪轻轻上下吸动打匀，使细胞重新悬浮；
(9)4℃下3000g冷冻离心5min；
(10)加入20μL冰冷10％的甘油，用移液器轻轻上下吸动打匀，使细胞重新悬浮；
(11)立即使用或迅速置于-70℃超低温保存。
4.单加氧酶活性检测
10000rpm离心10min，收集菌体细胞；加入50mM磷酸盐缓冲液(pH7.0)洗涤菌体2次，重悬于含有50mM吲哚的磷酸盐缓冲液(pH7.0)；将20mL重悬菌液转移至50mL离心管中，置于30℃水浴中150rpm孵育30min，每5min检测吲哚的消耗量。
单加氧酶酶活定义：在30℃下，上述反应体系中每1min消耗1μmol吲哚所需要的酶量为1个酶活单位(U)。
酶活检测结果表明，styAB基因超量表达的重组菌株B4-01在4h、12h和20h均表现出了显著增强的单加氧酶活性，约为对照菌株B4-0和野生型菌株B4的5倍(见图2)。
5.发酵试验
发酵培养基(1L)：17.1gNa2HPO4.12H2O，0.3gKH2PO4，0.1gNH4Cl，0.05gNaCl，0.01gMgSO4，0.1g酵母提取物。
挑取新鲜培养的Pseudomonasputida菌株接种于LB培养基中，置于恒温箱中30℃，200rpm培养24h进行活化。将新鲜活化的种子培养物转接至装有100mL发酵培养基的500mL锥形瓶中，30℃，200rpm进行发酵培养，依次在培养4、8、12、24、36和48h时收集发酵液。
6.靛蓝的检测
9500rpm离心10min收集50mL发酵液中的蓝色沉淀颗粒，无菌水洗涤后加入二甲基甲酰胺重悬靛蓝色素。将重悬液反复超声处理5min，过滤待测。
采用Agilent1290高效液相色谱仪检测靛蓝，检测条件为：C18色谱柱，二极管阵列检测器，流动相V(甲醇)∶V(超纯水)＝90∶10，柱温30℃，流速0.2mL/min，检测波长610nm。
检测结果表明，发酵24h后，各菌株中靛蓝色素产量达到峰值，重组菌株B4-01中靛蓝色素的含量最高，为52.13mg/L，是对照菌株B4-0和野生型菌株B4中靛蓝产量的7倍(见图3)。

Claims (5)

1.一株产靛蓝色素的重组Pseudomonasputida基因工程菌株的构建方法，其特征为携带单加氧酶基因styAB超量表达的游离型表达载体。
2.权利要求1中所述的构建方法运用的新设计思路，即内源苯乙烯单加氧酶基因styAB在Pseudomonasputida中的超量表达及其对靛蓝色素合成量的显著提高作用。
3.权利要求1中所述的Pseudomonasputida为PseudomonasputidaB4，其特征为野生型菌株含有内源苯乙烯单加氧酶基因styAB。
4.扩增权利要求3中所述苯乙烯单加氧酶基因styAB的特异性引物，分别为：
上游引物：5’-CGGGGTACCAGCAACCCACAACAAAAACAAG-3’
下游引物：5’-CCCAAGCTTGTGATCGGCACAGAAAGG-3’
5.权利要求1中所述的表达载体为pBBR1MCS-2-styAB，其特征为携带Pseudomonasputida内源苯乙烯单加氧酶基因styAB，该载体还包含以下元件：Pseudomonas启动子、转录终止子、复制蛋白Rep、卡那霉素抗性基因Km。
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US10975243B2 (en) 2018-12-28 2021-04-13 Industrial Technology Research Institute Genetically modified microorganism and method for producing indigo dye
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CN113604413A (zh) * 2021-07-22 2021-11-05 北京工商大学 一种重组菌株及制备方法与应用

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US10975243B2 (en) 2018-12-28 2021-04-13 Industrial Technology Research Institute Genetically modified microorganism and method for producing indigo dye
CN113564189A (zh) * 2021-06-11 2021-10-29 大连理工大学 一种高产靛蓝的工程菌制备靛蓝及其染布的方法
CN113604413A (zh) * 2021-07-22 2021-11-05 北京工商大学 一种重组菌株及制备方法与应用
CN113604413B (zh) * 2021-07-22 2023-06-20 北京工商大学 一种重组菌株及制备方法与应用

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