CN107287144A - 一种代谢改造的枯草芽孢杆菌生物转化细胞及其制备方法与应用 - Google Patents
一种代谢改造的枯草芽孢杆菌生物转化细胞及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种代谢改造的枯草芽孢杆菌生物转化细胞,所述枯草芽孢杆菌生物转化细胞的制备方法为:首先经过重组过表达获得一种L‑谷氨酸氧化酶,然后再对枯草芽孢杆菌内源谷氨酸分解途径进行代谢工程改造,最后改造了枯草芽孢杆菌对谷氨酸转运途径,制得所述枯草芽孢杆菌生物转化细胞,即重组枯草芽孢杆菌。本发明为参与细胞内源代谢的全细胞转化系统的构建及改良提供了实验依据,为利用枯草芽孢杆菌全细胞高效转化L‑谷氨酸生产α‑酮戊二酸提供借鉴。
Description
技术领域
本发明涉及基因工程代谢改造及微生物技术领域,尤其是涉及一种含有L-谷氨酸氧化酶基因的重组枯草芽孢杆菌及其制备方法。
背景技术
生物转化是指利用生物学的方法合成有机化合物的过程,即利用全细胞或提取酶作为催化剂将底物转化成目标产物的过程。
生物转化实际是利用有活性的酶,因此,其催化的反应具有如下特征:反应的专一性,立体的选择性,且反应条件要求不高,与传统的化学合成方法相比,生物催化的方法可以在低污染、低能耗、高特异性的条件下进行,它可以替换多步化学反应成一步酶催化反应或是引入化合物结构的多样性,所以目前广泛应用在有机溶剂、聚合物材料、医药工业中间体、有机化学光学对映体、抗生素、维生素等重要工业产品的生产中。越来越多的微生物资源被报道应用于生物转化,其研究思路的发展历程,经过了从利用野生型全细胞进行催化,到利用基因工程表达重组酶用来转化,最后进入了组合生物转化和代谢工程的全局性调控阶段。由于具有方向性强、目标明确、可控性好和重现性好的优势,代谢工程在生物转化宿主菌的改造方面具有良好的应用前景,合成生物学的出现又将这种应用提升到了一种多元化的新高度。
全细胞催化由于多样性和易操作性的优点在工业上得到了迅速的应用,但是也存在以下几方面的问题:底物跨膜的通透性大小影响最终的转化率;副反应导致底物或产物的降解;存在旁路反应和副产物积累的问题。这些问题一定程度上限制了全细胞转化在工业上的应用,因此利用基因工程异源表达重组酶或对天然酶进行定向改造在生物转化领域得到了迅速的发展。
枯草芽孢杆菌是在工业发酵和微生物分子遗传领域具有重要价值的革兰氏阳性菌,构建枯草芽孢杆菌基因工程菌的最主要方法是利用能独立复制的质粒对该菌进行遗传改造。枯草芽孢杆菌具有发酵周期短、产物丰富、可利用开发价值高以及作为食品药品安全性好等显著特点,它可产生多种抗生素,包括脂肽类、肽类、磷脂类、多烯类、氨基酸类、核酸类物质,对多种动、植物及人类病原菌起到很好的抑制作用。而且芽孢杆菌还具有很强的蛋白酶、脂肪酶、淀粉酶活性。因此,芽孢杆菌被广泛应用于医药、农药、食品、饲料加工、环境污染治理等各个行业。据不完全统计枯草芽孢杆菌所产的酶占整个酶市场的50%,是工业上生产酶应用最广泛的菌种之一。但目前对利用重组改造后的芽孢杆菌进行全细胞转化实验研究鲜有报道。
发明内容
针对现有技术存在的上述问题,本发明申请人提拱了一种代谢改造的枯草芽孢杆菌生物转化细胞及其制备方法与应用。本发明首先制备了含有密码子优化后的L-谷氨酸氧化酶基因的重组枯草芽孢杆菌,接着,经代谢工程改造宿主的谷氨酸分解途径和其转运途径,实现重组菌的全细胞转化L-谷氨酸生产α-酮戊二酸。本发明为参与细胞内源代谢的全细胞转化系统的构建及改良提供了实验依据,为利用枯草芽孢杆菌全细胞高效转化L-谷氨酸生产α-酮戊二酸提供借鉴。
本发明的技术方案如下:
一种代谢改造的枯草芽孢杆菌生物转化细胞,所述枯草芽孢杆菌生物转化细胞的制备方法为:首先经过重组过表达获得一种L-谷氨酸氧化酶,然后再对枯草芽孢杆菌内源谷氨酸分解途径进行代谢工程改造,最后改造了枯草芽孢杆菌对谷氨酸转运的途径,制得所述枯草芽孢杆菌生物转化细胞,即重组枯草芽孢杆菌。
编码所述L-谷氨酸氧化酶的氨基酸序列如SEQ ID No.1所示;所述L-谷氨酸氧化酶的基因序列如SEQ ID No.2所示。
所述枯草芽孢杆菌代谢工程改造的过程为:
(1)获取链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)所得L-谷氨酸氧化酶基因LGOX克隆至含有木糖的诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)将步骤(2)所得重组质粒pHY-Bs.xyl-LGOX转化至枯草芽孢杆菌WB600中,获得重组枯草芽孢杆菌WB600/pHY-Bs.xyl-LGOX,即重组枯草芽孢杆菌WB602。
所述枯草芽孢杆菌代谢工程改造的过程还可以为:
(1)获取链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)中所得L-谷氨酸氧化酶基因LGOX克隆至木糖诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)敲除枯草芽孢杆菌WB600中的谷氨酰胺合酶基因glnA,同时实现步骤(1)所述L-谷氨酸氧化酶基因的整合,构建了重组枯草芽孢杆菌WB603;
(4)将步骤(2)所得重组质粒pHY-Bs.xyl-LGOX转化至重组枯草芽孢杆菌WB603中,获得重组枯草芽孢杆菌WB603/pHY-Bs.xyl-LGOX,即代谢改造的重组枯草芽孢杆菌WB604。
所述枯草芽孢杆菌代谢工程改造的过程还可以为:
(1)获取来自链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)中所得L-谷氨酸氧化酶基因LGOX克隆至木糖诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)PCR扩增来源于Bacillus subtilis 168的谷氨酸转运蛋白GltP基因gltP;
(4)将步骤(3)扩增后的基因gltP克隆至步骤(2)所获得的重组质粒pHY-Bs.xyl-LGOX上,构建重组共表达质粒pHY-Bs.xyl-LGOX-GltP;
(5)将步骤(4)所得的重组共表达质粒pHY-Bs.xyl-LGOX-GltP转化至枯草芽孢杆菌WB600中,获得重组枯草芽孢杆菌WB600/pHY-Bs.xyl-LGOX-GltP,即重组枯草芽孢杆菌WB605。
一种所述枯草芽孢杆菌生物转化细胞的应用,所述枯草芽孢杆菌生物转化细胞可用于全细胞转化L-谷氨酸生产α-酮戊二酸,提高α-酮戊二酸的转化效率。
所述枯草芽孢杆菌菌种的种子培养基及发酵培养基:种子培养基(g·L-1):酵母粉5,蛋白胨10,氯化钠10;发酵培养基(g·L-1):甘油5,酵母粉24,蛋白胨12,K2HPO4 12.54,KH2PO4 2.31,调节pH至7.0。
将重组菌经种子培养基活化12h后,转接5%的接种量至发酵培养基,至菌体达生长对数中期OD600约为5左右,开始补加终浓度为10g·L-1的木糖进行诱导,共发酵约20h,离心收集菌体,并用0.1mol·L-1的pH 7.0磷酸钠盐缓冲液洗涤菌体2次,进行全细胞转化。
转化条件:全细胞催化剂和底物L-谷氨酸若干,1200U·mL-1过氧化氢酶和100mmol·L-1pH7.0磷酸盐缓冲液,于10ml反应体系中,37℃,200r·min-1反应24h,离心,取上清煮沸20min,再离心,HPLC检测上清中L-谷氨酸与α-酮戊二酸的量。
本发明有益的技术效果在于:
本发明通过将目的基因整合至glnA相应位点,实现了glnA基因的敲除和目的基因的整合,提高了L-谷氨酸的有效转化率。本发明证实敲除细菌底物消耗的旁路代谢通道能有效提高转化率,推测敲除产物的消耗途径亦可提高目标产物量。同时,本发明在全细胞催化过程中过表达底物转运蛋白,提高了转化效率,为参与细胞内源代谢的全细胞转化系统的构建及改良提供了实验依据,及工业上进行全细胞高效催化生产α-酮戊二酸提供借鉴。
附图说明
图1为重组诱导型表达载体pHY-Bs.xyl-LGOX双酶切验证;
图2为基因突变敲除盒pMDGANPL酶切验证;
图3为Bacillus subtilis中glnA基因敲除PCR验证;
图4为共表达载体pHY-Bs.xyl-LGOX-GltP酶切验证;
图5为重组菌WB602与WB605细胞转化比较;
具体实施方式
下面结合附图和实施例,对本发明进行具体描述。
实施例1 重组枯草芽孢杆菌的构建:
经密码子优化合成来源于链霉菌(Streptomyces sp.X-119-6)的L-谷氨酸氧化酶(LGOX)基因序列(L-谷氨酸氧化酶的氨基酸序列如SEQ ID No.1所示;所述L-谷氨酸氧化酶的基因序列如SEQ ID No.2所示),并在5’端和3’端分别加上BamH Ⅰ和EcoR Ⅰ酶切位点,由公司合成上述LGOX基因并克隆至pUC57载体上,获得重组质粒pUC57-LGOX,接着,经分子克隆将上述LGOX基因连接至pHY-Bs.xyl诱导型表达载体,制得含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX,经BamH Ⅰ和EcoR Ⅰ双酶切验证(图1)获得相应大小的目的片段。
将未含外源LGOX基因的诱导型表达质粒pHY-Bs.xyl和重组质粒pHY-Bs.xyl-LGOX转化至枯草芽孢杆菌WB600中,获得重组菌WB600/pHY-Bs.xyl,WB600/pHY-Bs.xyl-LGOX,分别命名为WB601,WB602。
实施例2基因glnA的敲除及目的基因的整合
(1)以引物GlnA-F和GlnA-R分别为上下游引物经PCR扩增来自枯草芽孢杆菌B.subtilis WB600的基因glnA(谷氨酰胺合成酶),经T-A克隆连接至pMDT19-simple质粒载体,构建重组质粒,分别标记为pMD-glnA,在上述扩增的基因中分别选取两个合适的酶切位点Nco Ⅰ和Pst Ⅰ,双酶切后,去除818bp的部分glnA基因片段,胶回收获得3210bp的片段,即以剩余部分为同源臂;
(2)以B.subtilis WB600基因组为模板,P43-Nco Ⅰ-F,P43-R-LGOX为上下游引物扩增大小为426bp的P43启动子,以pUC57-LGOX为模板,P43-LGOX-F,LGOX-Pst Ⅰ-R为上下游引物扩增LGOX基因,以上述扩增出的LGOX和P43为模板,P43-Nco Ⅰ-F,LGOX-Pst Ⅰ为上下游引物,将L-谷氨酸氧化酶基因与P43启动子进行融合PCR,实现两者的无缝连接,将融合好的片段经Nco Ⅰ和Pst Ⅰ双酶切,连接至经相同酶切的pMD-glnA质粒载体,构建重组质粒载体,记为pMD-glnA-P43-LGOX,测序融合部分P43-LGOX;
(3)经PCR扩增来源于PMA5质粒载体上的Neo(新霉素基因),并在引物两端分别加上与P43启动子上游相同的酶切位点,连接至T载体,测序,Nco Ⅰ酶切胶回收目的片段Neo,与步骤(2)中所获得的重组质粒载体pMD-glnA-P43-LGOX经相同酶切后,连接,构建重组质粒载体(图2),记为pMD-glnA-Neo-P43-LGOX,至此基因突变敲除盒pMDGANPL构建成功;
(4)将构建好的glnA和amyE基因敲除突变盒pMDGANPL经枯草芽孢杆菌化转至B.subtilis WB600,涂布含有6μg·mL-1的新霉素平板,37℃,培养16~24h后,挑取单菌落用表1中敲除验证引物GlnA-QCYZ-F,P43-R-LGOX进行PCR鉴定(图3),若glnA基因敲除成功,则PCR产物片段大小为1727bp,敲除后的突变菌命名为WB603,而基因未成功敲除则无法得到PCR产物片段。本实施例中所用引物序列如表1所示。
表1
注:上表中“单下划线”为酶切位点,“双下划线为”终止密码子
实施例3重组菌株WB604的构建
按照化转的方法将表达质粒pHY-Bs.xyl-LGOX转化至WB603,构建重组菌WB604,具体实施方式如下:
培养基:枯草芽孢杆菌B.subtilis WB600转化用培养基:
SP Ⅰ-a溶液(g·L-1):(NH4)2SO4 4,K2HPO4·3H2O 28,KH2PO4 12,二水合柠檬酸钠2;
SP Ⅰ-b溶液(g·L-1):MgSO4·7H2O 0.4;
500g·L-1葡萄糖溶液;
100×CAYE溶液(g·L-1):酪蛋白氨基酸20,酵母粉100;
CaCl2溶液:50mmol·L-1;
MgCl2溶液:250mmol·L-1;
100×EGTA溶液:称取3.8g EGTA(乙二醇双四乙酸)溶解于1L去离子水中,并用NaOH调pH至8.0,过滤除菌,-20℃保存备用;
将1~6中所配试剂于115℃高温高压灭菌20min,冷却后放置4℃冰箱备用;
用以上溶液配制枯草芽孢杆菌转化用培养基(现配现用):
SP Ⅰ培养基(20mL):SP Ⅰ-a和SP Ⅰ-b各9.8mL,50%葡萄糖溶液和100×CAYE溶液各200μL,混匀;
SP Ⅱ培养基(6mL):5.88mL SP Ⅰ培养基,60μL CaCl2溶液,60μL MgCl2溶液,混匀,向50mL离心管中每管分装2mL,备用。
B.subtilis WB600感受态细胞的制备与转化
(1)菌种活化,从保藏管中接种50μL菌株至3mL SP Ⅰ培养基中,37℃,200r·min-1过夜培养;
(2)从上述3mL SP Ⅰ培养基中转接100μL于5mL SP Ⅰ培养基中,37℃,200r·min-1培养4.5h;
(3)从5mL SP Ⅰ培养基中转接200μL于2mL SP Ⅱ培养基中,37℃,200r·min-1培养1.5h;
(4)向2mL SP Ⅱ培养基中加入20μL 100×EGTA溶液,37℃,200r·min-1培养10min;
(5)将上述菌悬液分装至1.5mL离心管中,每管500μL,直接用于转化或-70℃保存备用;
转化:
(1)将连接产物或质粒加入制备好的感受态中,混匀,37℃培养2.5h;
(2)5000r·min-1离心后培养的菌体2min,弃部分上清,用移液枪吹吸重悬菌体涂布于抗性平板;
(3)涂布抗性筛选平板于37℃过夜培养,挑取单菌落进行PCR及酶切验证。
实施例4 全细胞催化剂的制备及转化实验研究
将上述构建好的4株重组菌WB601,WB602,WB603,WB604按5%接种量分别接种于发酵培养基发酵,8h后补加10g·L-1的木糖进行诱导发酵共20h,离心收集、洗涤菌体两次,进行全细胞转化实验。
转化条件:2.5g·L-1 DCW全细胞催化剂,10g·L-1的L-谷氨酸底物,1200U·mL-1过氧化氢酶和100mmol·L-1pH7.0磷酸盐缓冲液,于10ml反应体系中,37℃,200r·min-1反应24h,离心,检测上清中L-谷氨酸与α-酮戊二酸的量。
转化结果如表2,通过WB601,WB603两株重组菌比较,仅实现LGOX基因整合的突变菌WB603的α-KG产量从原始对照菌WB601的0.08g·L-1提高到0.32g·L-1,表明将LGOX基因整合至染色体上可以使重组菌获得一定酶活,将L-谷氨酸转化生成少量α-KG,但由于拷贝数过低,致使表达量不高,不适合直接进行全细胞转化实验,故在此基础上再导入一游离质粒pHY-Bs.xyl-LGOX,构建了相应的重组菌WB604与WB602比较,转化率从glnA基因敲除前的87.9%提高至95.4%,说明glnA基因的表达会消耗部分底物,从而降低生物转化率,而本发明通过将目的基因整合至glnA相应位点,实现了glnA基因的敲除和目的基因的整合,使其转化率相应提高。本实验证实敲除细菌底物消耗的旁路代谢通道能有效提高转化率,推测敲除产物的消耗途径亦可提高目标产物。
表2
实施例5 共表达载体的构建
提取枯草芽孢杆菌染色体为模板,以表1中GltP-Xba Ⅰ-F和GltP-Xba Ⅰ-R为上下游引物,扩增完整的转运蛋白GltP片段(1396bp),经TA克隆、转化等操作连接至pMDT-19Simple载体,构建质粒T-GltP,送至上海生工生物工程有限公司测序验证正确,此质粒经Xba Ⅰ酶切,割胶回收1396bp目的片段,连接至经相同酶切的诱导型穿梭质粒载体pHY-Bs.xyl-LGOX,构建了重组质粒pHY-Bs.xyl-LGOX-GltP,分别经BamH Ⅰ和EcoR Ⅰ双酶切,XbaⅠ单酶切,分别获得7648bp和2103bp(LGOXstr基因片段),8355bp和1396bp(GltP基因片段)的DNA条带,如图4所示,所得条带与DNA序列分析结果一致,说明重组质粒构建成功。
实施例6 共表达菌株的构建及细胞转化
将构建好的重组质粒载体pHY-Bs.xyl-LGOX-GltP转化至WB600,构建重组菌WB600/pHY-Bs.xyl-LGOX-GltP,命名为WB605。活化种子WB602和WB605,将重组菌WB602与WB605进行全细胞转化L-谷氨酸实验。结果发现(图5):在40g·L-1L-谷氨酸,1g·L-1葡萄糖,1U·mL-1的全细胞催化剂酶活力的条件下转化30h,定点取样,发现表达了转运蛋白GltP的重组菌WB605在20h时,底物也基本消耗完,产生37.8g·L-1的α-酮戊二酸,达到95.1%的转化率,此段时间内的平均转化速率达1.89g·L-1·h-1;而未表达转运蛋白的重组菌WB602在转化20h时,产生约30.9g·L-1的α-酮戊二酸,其转化率为77.8%,平均转化速率为1.55g·L-1·h-1;转运蛋白GltP的过表达使得达平衡期时的全细胞平均转化速率提高了21.9%。说明转运蛋白GltP的过表达,加速了细胞将L-谷氨酸转运至细胞内的速率,与未表达转运蛋白GltP重组菌相比,在LGOX酶活力相同的条件下,提高了谷氨酸的运输速率,从而提高了转化效率。
Claims (6)
1.一种代谢改造的枯草芽孢杆菌生物转化细胞,其特征在于所述枯草芽孢杆菌生物转化细胞的制备方法为:首先经过重组过表达获得一种L-谷氨酸氧化酶,然后再对枯草芽孢杆菌内源谷氨酸分解途径进行代谢工程改造,最后改造了枯草芽孢杆菌对谷氨酸转运的途径,制得所述枯草芽孢杆菌生物转化细胞,即重组枯草芽孢杆菌。
2.根据权利要求1所述的枯草芽孢杆菌生物转化细胞,其特征在于编码所述L-谷氨酸氧化酶的氨基酸序列如SEQ ID No.1所示;所述L-谷氨酸氧化酶的基因序列如SEQ ID No.2所示。
3.根据权利要求1所述的枯草芽孢杆菌生物转化细胞,其特征在于所述枯草芽孢杆菌代谢工程改造的过程为:
(1)获取链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)所得L-谷氨酸氧化酶基因LGOX克隆至含有木糖的诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)将步骤(2)所得重组质粒pHY-Bs.xyl-LGOX转化至枯草芽孢杆菌WB600中,获得重组枯草芽孢杆菌WB600/pHY-Bs.xyl-LGOX,即重组枯草芽孢杆菌WB602。
4.根据权利要求1所述的枯草芽孢杆菌生物转化细胞,其特征在于所述枯草芽孢杆菌代谢工程改造的过程为:
(1)获取链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)中所得L-谷氨酸氧化酶基因LGOX克隆至木糖诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)敲除枯草芽孢杆菌WB600中的谷氨酰胺合酶基因glnA,同时实现步骤(1)所述L-谷氨酸氧化酶基因的整合,构建了重组枯草芽孢杆菌WB603;
(4)将步骤(2)所得重组质粒pHY-Bs.xyl-LGOX转化至重组枯草芽孢杆菌WB603中,获得重组枯草芽孢杆菌WB603/pHY-Bs.xyl-LGOX,即代谢改造的重组枯草芽孢杆菌WB604。
5.根据权利要求1所述的枯草芽孢杆菌生物转化细胞,其特征在于所述枯草芽孢杆菌代谢工程改造的过程为:
(1)获取来自链霉菌(Streptomyces sp.X-119-6)来源,经密码子优化后的L-谷氨酸氧化酶基因LGOX;
(2)将步骤(1)中所得L-谷氨酸氧化酶基因LGOX克隆至木糖诱导型表达载体pHY-Bs.xyl上,构建含有L-谷氨酸氧化酶基因的重组质粒pHY-Bs.xyl-LGOX;
(3)PCR扩增来源于Bacillus subtilis 168的谷氨酸转运蛋白GltP基因gltP;
(4)将步骤(3)扩增后的基因gltP克隆至步骤(2)所获得的重组质粒pHY-Bs.xyl-LGOX上,构建重组共表达质粒pHY-Bs.xyl-LGOX-GltP;
(5)将步骤(4)所得的重组共表达质粒pHY-Bs.xyl-LGOX-GltP转化至枯草芽孢杆菌WB600中,获得重组枯草芽孢杆菌WB600/pHY-Bs.xyl-LGOX-GltP,即重组枯草芽孢杆菌WB605。
6.一种权利要求1~5任一项所述枯草芽孢杆菌生物转化细胞的应用,其特征在于所述枯草芽孢杆菌生物转化细胞可用于全细胞转化L-谷氨酸生产α-酮戊二酸,提高α-酮戊二酸的转化效率。
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