CN112111407A - 一种生产碱性果胶裂解酶的集胞藻pcc6803藻株及其构建方法 - Google Patents
一种生产碱性果胶裂解酶的集胞藻pcc6803藻株及其构建方法 Download PDFInfo
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Abstract
本发明公开了一种生产碱性果胶裂解酶的集胞藻PCC6803藻株及其构建方法,属于工业微生物领域。本发明通过同源重组的方法将来源于克雷伯菌属(Klebsiella sp.Y1)的碱性果胶裂解酶基因petyl重组至集胞藻PCC6803基因组中,构建一种产碱性果胶裂解酶petyl的集胞藻PCC6803基因工程藻株PSY1,从而实现一种利用光合生物来生产果胶酶的方法,其不但获得可利用无机盐和二氧化碳来生产果胶酶的新途径,也降低了在生物质转化过程中能源(主要为碳源)的消耗。
Description
技术领域
本发明涉及一种生产碱性果胶裂解酶的集胞藻PCC6803藻株及其构建方法,属于工业微生物领域。
背景技术
随着全球人口密度的逐渐增大,能源短缺问题以及环境问题也越来越严峻,果胶裂解酶作为一种温和有效的催化剂,在生物质降解方面有巨大的潜力,其在工业生产中的应用也越来越广泛。植物细胞壁主要由多糖和蛋白质组成,部分含有木质素。细胞壁多糖主要分为纤维素、半纤维素、果胶质,纤维素位于半纤维素和果胶质形成的基质中。果胶质广泛存在于高等植物中,是植物细胞间质和初生细胞壁的重要组分,在植物细胞中起着粘合作用。果胶质是高分子聚合物,骨架为由α-1,4糖苷键连接的半乳糖醛酸残基,部分甲酯化,在富含聚L-鼠李糖区域有木聚糖和阿拉伯半乳聚糖侧链。作为一种结构复杂的大分子物质,果胶质的存在阻碍了我们对可再生生物质资源在食品、材料和能源方面的降解利用。能降解果胶质的复合酶统称为果胶酶。按照酶的作用方式,果胶酶可分为原果胶酶、果胶酯酶和解聚酶。原果胶酶可将不溶性果胶质分解为可溶性果胶质,降低果胶质粘度;果胶酯酶作用于C-6位甲酯键,使果胶中甲酯水解生成果胶酸,其最适底物为65%-75%甲酯化的果胶。解聚酶又包括水解酶和裂解酶。果胶水解酶通过水解作用切断果胶质中的α-1,4糖苷键,果胶裂解酶则主要通过β-消除反应作用于聚半乳糖醛酸的α-1,4糖苷键,在生成的寡聚半乳糖醛酸的非还原末端形成Δ4,5不饱和键。果胶酶按其适用的PH范围又可分为酸性果胶酶和碱性果胶酶。其作用的PH不同,应用的范围也不同。酸性果胶酶主要用于果汁及酒类的提取、澄清,并应用于饲料中消除饲料中的抗营养成分。碱性果胶酶近年来也受到广泛关注,在植物纤维和织物脱胶、咖啡和茶的发酵、油类提取、含果胶工业废水处理及造纸方面都有一定的应用。随着碱性果胶酶在轻工纺织业越来越受到重视,近年来国内外许多研究人员致力于对碱性果胶酶的挖掘和异源表达。国内外对产果胶酶的细菌菌种蹄选、工业发酵条件优化、基因工程方面都有研究。传统酶制剂主要是通过微生物的初级发酵产品来生产,需要通过各种理化因素的改变或对菌种进行突变,但往往提高产酶量的幅度有限。随着分子生物学和基因工程技术的迅猛发展,人们对果胶酶基因开始进行研究。近些年来,人们对碱性果胶裂解酶基因已通过基因工程技术得到了表达,这些表达宿主主要为E.coli、毕赤酵母。而这些异养微生物利用有机碳源如麸皮、玉米杆、稻草等有机碳源为原料进行生产,在酶生产过程中消耗了大量有机碳源,从能量转化及经济角度均不合理,严重限制了利用生物质进行生物能源转化的效率和可行性。如能通过改造光合生物,依靠光合作用,利用二氧化碳和简单无机盐为主的培养基来生产果胶酶,就可以减少果胶酶生产过程中的碳源和能源消耗,提高生物质转化的效率,不仅可以降低生物质转化过程的能源和碳源消耗,而且还可以实现对温室气体的有效利用。
蓝藻是一种能够利用ATP来固定大气CO2的产氧光合原核生物,其生长条件简单,只需在光照下利用一些简单营养物质便能生长。随着蓝藻基因工程的发展,藻类被越来越多地被用于基因工程的优良宿主。集胞藻(Synechocystis sp.PCC6803)是一种单细胞蓝藻,是第一个完成全基因组测序的蓝细菌,能进行产氧光合作用。用集胞藻PCC6803生产生物质酶不仅不需要有机碳源的消耗,同时也对减少环境污染及修复水体环境具有一定积极影响。
本发明为从克雷伯菌属(Klebsiella sp.Y1)中获得的外源基因碱性果胶裂解酶pety1基因通过基因重组技术整合至集胞藻基因组中,获得一种产碱性果胶裂解酶pety1的集胞藻PCC6803基因工程藻株PSY1,从而实现利用光合生物来获得果胶酶的方法,其降低了在生物质转化过程中能源(主要为碳源)的消耗。
发明内容
为了克服现有技术的缺点与不足,本发明目的在于提供一种可生产碱性果胶裂解酶pety1的集胞藻PCC6803工程藻株PSY1,利用光合生物获得果胶酶。
本方法还提供一种可生产碱性果胶裂解酶pety1的集胞藻工程藻株的构建方法。
本发明的技术方案如下:
上述一种可生产果胶酶pety1的集胞藻工程藻株的构建方法,步骤如下:
(1)以序列表中SEQ ID NO:1和SEQ ID NO:2为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得光感启动子兼上游臂psbA2-up基因片段,通过引物在3’端引入Hpa1酶切位点,获得的序列如序列表中SEQ ID NO:3所示;以序列表中SEQ ID NO:4和SEQ ID NO:5为上下游引物,以GenBank登录号为U02439.1的大肠杆菌为模板,通过pcr扩增技术获得大肠杆菌终止子T1T2基因片段,获得的序列如序列表中SEQ ID NO:6所示;以序列表中SEQ ID NO:7和SEQ ID NO:8为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得下游臂downstream基因片段,获得的序列如序列表中SEQ ID NO:9所示;
(2)将步骤(1)获得的大肠杆菌终止子T1T2基因片段进行PStI和BamHI双酶切,与经过相同双酶切的pBluescript SK质粒通过T4连接酶连接,得到质粒pBluescript SKT1T2;
(3)将步骤(1)获得的downstream基因片段用SacI与Sac II双酶切后,与经相同酶切的质粒pBluescriptSKT1T2连接,得到质粒pBluescript SK T1T2-downstream;
(4)用限制性内切酶BamHI分别单酶切pBluescript SK T1T2-downstream与pUC4K质粒,电泳后分别胶回收pBluescript SK T1T2-downstream载体片段和kana抗性基因片段。对载体片段经磷酸化处理后与kana抗性基因片段连接,得到质粒pBluescript SKT1T2-npt-downstream;
(5)用限制性内切酶ApaI处理质粒pBluescript SK T1T2-npt-downstream,获得pBluescript SK T1T2-npt-downstream载体片段,对载体片段经磷酸化处理后与步骤(1)中的psbA2-up基因片段进行同源重组,获得质粒p5st1t2npt,如图1所示;
(6)以序列表中SEQ ID NO:10和SEQ ID NO:11为上下游引物,以GenBank数据库中克雷伯菌属(Klebsiella sp.Y1)的相应基因序列为模板,通过pcr扩增技术获得碱性果胶裂解酶基因片段pety1,如图2所示,序列如序列表中SEQ ID NO:12所示;
(7)用限制性内切酶HpaI单酶切质粒p5st1t2npt,获得p5st1t2npt载体片段,对载体片段经磷酸化处理后与步骤(6)中的pety1基因片段进行同源重组,获得重组表达质粒PSTY1,如图3所示;
(8)将步骤(7)所得的重组表达载体质粒PSTY1经自然转化至集胞藻PCC6803中,经过抗性筛选,获得转基因集胞藻,即PSTY1工程藻株;
一种可生产碱性果胶裂解酶的集胞藻PCC6803藻株通过上述方法构建得到。
附图说明
图1为同源重组质粒p5st1t2npt结构图
图2为碱性果胶裂解酶基因片段pety1电泳图
图3为重组表达载体PSTY1结构图
图4为D-半乳糖醛酸标准曲线图
图5为工程藻株与野生藻株酶活图
本发明通过同源重组技术将外源基因pety1整合至集胞藻PCC6803基因组上,通过此方法可使集胞藻PCC6803工程藻株产碱性果胶裂解酶,通过果胶酶酶活测定,工程藻株酶活优于野生藻株。本发明的可生产碱性果胶裂解酶工程藻株对构建生产果胶酶的基因工程菌具有重要的理论意义,对生物质转化具有重要的现实意义。
具体实施方式
下面结合说明书附图及实施例对本发明的技术方案做进一步说明,但本发明所保护范围不限于此。
实施例1
p5st1t2npt质粒的构建
以序列表中SEQ ID NO:4和SEQ ID NO:5为上下游引物,以GenBank登录号为U02439.1的大肠杆菌为模板,通过pcr扩增技术获得大肠杆菌终止子T1T2基因片段,获得的序列如序列表中SEQ ID NO:6所示。扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,40s,30个循环;72℃,5min,4℃,10min。pcr反应结束后进行电泳切胶回收。将获得的大肠杆菌终止子T1T2基因片段进行PStI和BamHI双酶切,与经过相同双酶切的pBluescript SK质粒通过T4连接酶连接,转化大肠杆菌DH5α,筛选阳性克隆并测序,得到质粒pBluescript SKT1T2。酶切体系为:DNA 3μL,内切酶各1μL,10×Buffer1μL,ddH2O 4μL,酶切温度为30℃,酶切时间为2h。以序列表中SEQ ID NO:7和SEQ ID NO:8为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得下游臂downstream基因片段,获得的序列如序列表中SEQ ID NO:9所示。扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,1.5min,30个循环;72℃,5min,4℃,10min。pcr反应结束后进行电泳切胶回收。将获得的downstream基因片段用SacI与Sac II双酶切后,与经相同酶切的质粒pBluescript SK T1T2连接,转化大肠杆菌DH5α,筛选阳性克隆并测序,得到质粒pBluescript SK T1T2-downstream。酶切体系为:DNA3μL,内切酶各1μL,10×Buffer1μL,ddH2O 4μL,酶切温度为30℃,酶切时间为2h。用限制性内切酶BamHI分别单酶切pBluescript SK T1T2-downstream与pUC4K质粒,电泳后分别胶回收pBluescript SK T1T2-downstream载体片段和kana抗性基因片段。对载体片段经磷酸化处理后与kana抗性基因片段连接,转化大肠杆菌DH5α,筛选阳性克隆并测序,得到质粒pBluescript SK T1T2-npt-downstream。酶切体系为:DNA 3μL,内切酶2μL,10×Buffer1μL,ddH2O 4μL,酶切温度为30℃,酶切时间为2h。以上连接体系为:回收获得的T1T2基因片段0.5μL,回收处理的质粒片段2.5μL,T4连接酶1μL,10×Buffer 1μL,ddH2O 5μL,总体系10μL,反应温度为16℃,连接10h。
以序列表中SEQ ID NO:1和SEQ ID NO:2为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得光感启动子兼上游臂psbA2-up基因片段,通过引物在3’端引入Hpa1酶切位点,获得的序列如序列表中SEQ ID NO:3所示。扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,30s,30个循环;72℃,5min,4℃,10min。pcr反应结束后进行电泳切胶回收。用限制性内切酶ApaI单酶切质粒pBluescript SK T1T2-npt-downstream,经磷酸化处理后获得pBluescript SK T1T2-npt-downstream载体片段,与psbA2-up基因片段在重组酶作用下进行同源重组,转化大肠杆菌DH5α,筛选阳性克隆并测序,获得质粒p5st1t2npt。酶切体系为:DNA 3μL,内切酶2μL,10×Buffer1μL,ddH2O 4μL,酶切温度为37℃,酶切时间为2h。重组连接体系为:回收获得的psbA2-up基因1μL,ApaI处理后的pBluescript SK T1T2-npt-downstream载体片段2.5μL,重组酶2μL,10×Buffer 4μL,ddH2O 10μL,总体系20μL,反应温度37℃,反应时间40min。
实施例2
重组表达载体PSTY1的构建
以序列表中SEQ ID NO:10和SEQ ID NO:11为上下游引物,以GenBank数据库中克雷伯菌属(Klebsiella sp.Y1)的相应基因序列为模板,通过pcr扩增技术获得碱性果胶裂解酶基因片段pety1,序列如序列表中SEQ ID NO:12所示。扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,2min,30个循环;72℃,5min,4℃,10min。用限制性内切酶HpaI单酶切质粒p5st1t2npt,获得p5st1t2npt载体片段,对载体片段经磷酸化处理后与pety1基因片段在重组酶作用下进行同源重组,转化大肠杆菌DH5α,筛选阳性克隆并测序,获得重组表达载体PSTY1。酶切体系为:DNA 3μL,内切酶2μL,10×Buffer1μL,ddH2O 4μL,酶切温度为37℃,酶切时间为2h。重组连接体系为:回收获得的pety1基因1μL,经磷酸化处理后的p5st1t2npt载体片段2.5μL,重组酶2μL,10×Buffer 4μL,ddH2O 10μL,总体系20μL,反应温度37℃,反应时间40min。
实施例3
集胞藻PCC6803基因工程藻株PSTY1的获得及扩大培养
通过自然转化的方法将重组表达载体PSTY1重组至集胞藻PCC6803基因组中,经抗生素筛选,得到工程藻株PSTY1。具体步骤为:取对数培养期(OD730=0.6)的集胞藻PCC680350ml于室温,5000r/min离心10min,弃上清,加新鲜BG-11液体培养基洗涤一次后,加新鲜BG-11液体培养基至终浓度OD730=4.8,并立刻用来转化。将收集的藻液分装到1.5mlEP管中(每管400ul),每管加10ug PSTY1质粒,弱光条件下光照温育6小时,期间摇晃一次。将混合液涂到含有卡那霉素抗性(50μg/mL)的BG11平板培养基上,约10天可见转化子。将长出的藻落转移至含有相同浓度Kana抗生素的20mLBG-11小瓶培养基中培养,待长至对数期后转接培养,培养条件为28℃,1400Lux连续光照。
实施例4
野生集胞藻PCC6803藻株与工程藻株PSTY1的碱性果胶酶酶活测定
(1)粗酶液的制备
将野生型集胞藻PCC6803和工程藻株PSTY1接种于50mlBG-11液体培养基中,调节OD730=0.2,于28℃,1400Lux持续光照条件下培养至对数生长期,取30mL处于对数期的藻液,4℃、5000r/min离心10min,去上清,用10mL,50mmol/L pH7.0的PBS缓冲液重悬藻泥,4℃、5000r/min离心10min,去上清,收集藻细胞,收集的藻细胞经液氮研磨、超声破碎后制备成粗酶液。
(2)标准曲线的制备
果胶酶水解果胶,释放出还原性D-半乳糖醛酸,与3,5-二硝基水杨酸(即DNS)共热产生棕红色的氨基化合物,即发生显色反应。在一定范围内,还原糖的量和反应液的颜色呈比例关系,可利用比色法进行测定。通过分光光度计测定吸光度,与标准曲线比较,计算果胶酶酶活。酶活的定义为:在pH值为9.0,温度为50℃条件下,定义每分钟能催化分解果胶产生1μmol半乳糖醛酸的果胶酶量,为一个酶活力单位U。
式中:A-样品的吸光度
K-标准曲线斜率
b-标准曲线截距
n-酶液稀释倍数
t-反应时间/min
194.14-半乳糖醛酸的摩尔质量/(g/mol)
V-反应的体积/ml
准确称取D-半乳糖醛酸0.1000g,用pH值为5的柠檬酸-磷酸缓冲液定容于100ml容量瓶中,获得1.0mg/ml的D-半乳糖醛酸溶液。在25ml刻度试管中加入1.0mg/ml D-半乳糖醛酸和缓冲液,配制成一系列梯度浓度的D-半乳糖醛酸溶液,每个浓度设3个平行样,加入2.5mL DNS试剂,摇匀,沸水浴10min,冰浴30min。用酶标仪540nm测定吸收值,分别减去1管中的空白对照。以D-半乳糖醛酸浓度为横坐标,吸光度为纵坐标,制作标准曲线,得出曲线方程,如图4所示。
(3)酶活测定
在25ml试管中加入1ml 1%(w/v)果胶溶液,0.075ml 10mMCaCl2溶液,0.5ml粗酶液,50℃水浴1h,加2.5mlDNS试剂,煮沸10min。冰浴30min,用酶标仪540nm测定吸光度。酶活测定结果如图5所示。
由图5可以看出,集胞藻工程藻株PSTY1的酶活是野生集胞藻藻株酶活的3.6倍,结果证明利用同源重组技术将碱性果胶酶基因整合至集胞藻PCC6803基因组中,其工程藻株PSTY1的酶活高于野生集胞藻藻株酶活。
Claims (7)
1.一种生产碱性果胶裂解酶的集胞藻PCC6803的基因工程藻株及其构建方法。
2.如权利要求1所述的基因工程藻株,其构建方法包括如下步骤:
(1)以序列表中SEQ ID NO:1和SEQ ID NO:2为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得光感启动子兼上游臂psbA2-up基因片段,通过引物在3’端引入Hpa1酶切位点,获得的序列如序列表中SEQ ID NO:3所示;以序列表中SEQ ID NO:4和SEQ ID NO:5为上下游引物,以GenBank登录号为U02439.1的大肠杆菌为模板,通过pcr扩增技术获得大肠杆菌终止子T1T2基因片段,获得的序列如序列表中SEQ ID NO:6所示;以序列表中SEQ ID NO:7和SEQ ID NO:8为上下游引物,以野生集胞藻PCC6803基因组为模板,通过pcr扩增技术获得下游臂downstream基因片段,获得的序列如序列表中SEQ ID NO:9所示;
(2)将步骤(1)获得的大肠杆菌终止子T1T2基因片段进行PStI和BamHI双酶切,与经过相同双酶切的pBluescript SK质粒通过T4连接酶连接,得到质粒pBluescript SK T1T2;
将步骤(1)获得的downstream基因片段用SacI与Sac II双酶切后,与经相同酶切的质粒pBluescriptSKT1T2连接,得到质粒pBluescript SK T1T2-downstream;
用限制性内切酶BamHI分别单酶切pBluescript SK T1T2-downstream与pUC4K质粒,电泳后分别胶回收pBluescript SK T1T2-downstream载体片段和kana抗性基因片段。对载体片段经磷酸化处理后与kana抗性基因片段连接,得到质粒pBluescript SK T1T2-npt-downstream;
用限制性内切酶ApaI处理质粒pBluescript SK T1T2-npt-downstream,获得pBluescript SK T1T2-npt-downstream载体片段,对载体片段经磷酸化处理后与步骤(1)中的psbA2-up基因片段进行同源重组,获得质粒p5st1t2npt;
(3)以序列表中SEQ ID NO:10和SEQ ID NO:11为上下游引物,以GenBank数据库中克雷伯菌属(Klebsiella sp.Y1)的相应基因序列为模板,通过pcr扩增技术获得碱性果胶裂解酶基因片段pety1,序列如序列表中SEQ ID NO:12所示;
(4)用限制性内切酶HpaI单酶切质粒p5st1t2npt,获得p5st1t2npt载体片段,对载体片段经磷酸化处理后与步骤(3)中的pety1基因片段进行同源重组,获得重组表达质粒PSTY1;
(5)将步骤(4)所得的重组表达载体质粒PSTY1经自然转化至集胞藻PCC6803中,经过抗性筛选,获得转基因集胞藻,即PSTY1工程藻株。
3.如权利要求2所述的构建方法,其特征在于,所述步骤(1)中pcr扩增光感启动子兼上游臂psbA2-up基因片段引物为:
psbA2-up-F:5’-GATGTCGACGCTTTAGCGTTCCAGTG-3’;
psbA2-up-R:5’-CATTTGGTTATAATTCCTTATGTAT-3’;
扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,30s,30个循环;72℃,5min,4℃,10min。
4.如权利要求2所述的构建方法,其特征在于,所述步骤(1)中pcr扩增大肠杆菌终止子T1T2基因片段引物为:
T1T2-F:5’-ATACTGCAGCCAAGCTTGGCTGTTTTGGC-3’;
T1T2-R:5’-TTAGGATCCCCCATTATTGAAGCATTTAT-3’;
扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,40s,30个循环;72℃,5min,4℃,10min。
5.如权利要求2所述的构建方法,其特征在于,所述步骤(1)中pcr扩增下游臂downstream基因片段引物为:
downstream-F:5’-CTTCATATGCCGCGGATGACAACGACTCTCCAAC-3’;
downstream-R:5’-AGTGAGCTCTTAACCGTTGACAGCAGG-3’;
扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,1.5min,30个循环;72℃,5min,4℃,10min。
6.如权利要求2所述的构建方法,其特征在于,所述步骤(3)中pcr扩增碱性果胶裂解酶pety1基因片段引物为:
pety1-F:5’-AGGAATTATAACCAAGTTAACATGCAGGAGAACGAGCGCC-3’;
pety1-R:5’-CTCGAGGGGGGGCCCGTTAACTTAATGATGATGATGATGATGGCCGTTC-3’;
扩增程序为:94℃,3min;98℃,10s,55℃,15s,72℃,2min,30个循环;72℃,5min,4℃,10min。
7.根据权利要求1所述的生产碱性果胶裂解酶的集胞藻PCC6803藻株,其特征在于:所述的生产碱性果胶裂解酶的集胞藻PCC6803工程藻株,其工程藻株PSTY1的酶活高于野生集胞藻藻株酶活。
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CN114292866A (zh) * | 2022-01-06 | 2022-04-08 | 天津科技大学 | 一种生产木聚糖酶的集胞藻pcc6803的基因工程藻株、方法及应用 |
CN115772531A (zh) * | 2022-11-11 | 2023-03-10 | 吉林农业大学 | 一种果胶酸裂解酶基因突变体及其克隆方法和应用 |
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