CN111849852A - 一种高光学纯l-乳酸工程菌的构建方法 - Google Patents
一种高光学纯l-乳酸工程菌的构建方法 Download PDFInfo
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- CN111849852A CN111849852A CN202010936756.2A CN202010936756A CN111849852A CN 111849852 A CN111849852 A CN 111849852A CN 202010936756 A CN202010936756 A CN 202010936756A CN 111849852 A CN111849852 A CN 111849852A
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Abstract
本发明涉及一种高光学纯L‑乳酸工程菌的构建方法,包括多个步骤,本发明选取拟干酪乳杆菌(Lactobacillus paracasei NCBIO01)作为出发菌株,参照其全基因组序列,基于CRISPR/Cas9基因编辑方法构建了pNcasA‑△ldhD敲除质粒,无痕敲除D‑乳酸脱氢酶(ldhD)的同时过表达一个L‑乳酸脱氢酶基因(ldhL),得到一株高光学纯L‑乳酸工程菌,使其L‑乳酸光学纯度从原来的95.6%提高到99%以上,并且生产速率达到5.5g/L/h以上,成为潜在的工业L‑乳酸高效生产菌株。
Description
技术领域
本发明涉及乳酸菌技术领域,具体涉及一株高光学纯L-乳酸工程菌及其构建方法。
背景技术
乳酸作为一种多用途的天然有机酸,广泛应用于食品、日用化工、制药、纺织、环保和农业等诸多领域。乳酸的生产方法主要有微生物发酵法和化学合成法,微生物发酵生产乳酸具有成本低、光学纯度高、操作容易、安全性高以及污染小等优点,因此受到科研人员广泛关注。近年来,人们通过L-乳酸聚合生成聚L-乳酸(poly lactic acid,PLA),由于其生物相容性、可降解性以及无毒害作用等优良性能,能实现在生物圈中循环,是一种非常理想的高分子材料,在工业上具有广阔的应用前景。但是聚L乳酸的合成需要高光学纯度的L-乳酸作为前体物质,大多数同型乳酸发酵乳酸生产菌株具有较高的乳酸生产能力,但是会伴随着D-乳酸的生成,L-乳酸的光学纯度达不到合成聚乳酸的级别,必须通过下游的分离纯化步骤,加大了生产成本。
目前大部分乳酸生产菌株都是通过自然筛选和人工诱变获得,然而这些乳酸生产菌株在发酵过程中都存在一些限制性因素,例如同时存在L-乳酸脱氢酶和D-乳酸脱氢酶,产生L-乳酸和D-乳酸的异构体混合物,或者是异型乳酸发酵,乳酸光学纯度高,但副产物多,导致乳酸获得率低。随着分子生物学和基因组学、代谢组学的飞速发展,基因操作手段也越来越普及,人们可以通过对乳酸菌的定向基因编辑来获得所需性状的突变株。目前乳酸菌中应用最多的基因编辑方法是基于质粒的同源重组、单链DNA重组、Red/RecET介导的双链DNA重组等,但这些方法都操作复杂、耗时耗力,限制了乳酸菌代谢工程的发展,迫切地需要更高效更简便的方法。近年来,一种新型的基因编辑工具(CRISPR/Cas),被成功应用到哺乳动物、植物及微生物中,极大了促进了功能基因组学及基因疗法的发展。利用该方法可以对目标基因进行点突变、基因敲除及基因插入,并且能精准定位到基因组的目标位置。拟干酪乳杆菌是一株同型乳酸发酵生产L-乳酸的高产菌株,并且在发酵过程中不易受杂菌影响,操作简单,拥有较高的工业应用前景。传统基因编辑方法对拟干酪乳杆菌改造效率低下,其CRISPR基因编辑技术亦尚未有报道,若能成功开发将极大提高拟干酪乳杆菌基因组编辑效率,从而服务于高性能乳酸菌的遗传改造,并扩展产物的应用前景。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一株高光学纯L-乳酸工程菌的构建方法,其具有让L-乳酸光学纯度提高的优点。
为实现上述目的,本发明提供一种高光学纯L-乳酸工程菌的构建方法,包括如下步骤,
步骤1:制备PCR体系50μl,取PCR扩增产物,进行1%琼脂糖凝胶电泳鉴定,鉴定成功后对PCR产物进行纯化回收;
步骤2:根据拟干酪乳杆菌ldhD基因序列,在ldhD基因CDS区上游设计sgRNA,通过引物ldhDsg-F/ldhDsg-R PCR扩增得到sgRNA片段,以拟干酪乳杆菌基因组为模板,用引物ldhDup-F/ldhDup-R、ldhDdown-F/ldhDdown-R分别PCR扩增出ldhD上游同源臂和下游同源臂各1000bp,以ldhL-F/ldhL-R引物PCR扩增出L-乳酸脱氢酶基因(ldhL),以pan7-1质粒为模板,以Amp-F/Amp-R引物PCR扩增出氨苄青霉素抗性基因片段,
步骤3:再用PCR将氨苄青霉素抗性基因片段、上游同源臂、ldhL基因、下游同源臂以及sgRNA融合,引物设计时需要使融合的片段之间存在20-40bp的重复区,五个片段融合的摩尔比为1:3:5:3:1,得到构建完成的质粒pNcas-△ldhD;
步骤4:将保存好的Lactobacillus paracasei甘油管在一级MRS茄子瓶涂布活化,37℃培养36h。然后将一级MRS茄子瓶菌体转接到二级茄子瓶中,37℃静止培养18h。将二级茄子瓶中的菌体用50mL的无菌水洗涤,在MRS平板中划线,37℃静置培养36h,挑取单菌落接种至MRS液体培养基中,37℃,110r/min过夜培养,按照5%的比例转接至50mL含有1%浓度甘氨酸的MRS液体培养基中,37℃,110r/min培养至OD620值为0.6,然后4500rpm,4℃离心10min收集菌体,电转缓冲液洗两遍,用1mL预冷的电转缓冲液重悬菌体,按每管80μL分装于1.5mL离心管中,冻存于-80℃,取100ng的pNcas-△ldhD质粒,将其与感受态细胞充分混合均匀,转移至冰上预冷的2mm电极杯中,进行电转化,快速加入37℃预热的复苏液MMRS,转移至离心管中,37℃复苏培养3h,涂布抗性平板,37℃培养48h,计算其转化效率;
步骤5:用具有氨苄抗性的MRS平板筛选含有pNcas-△ldhD质粒的转化子,然后挑取转化子单菌落过夜复苏培养,提取突变株基因组用ldhD特异性引物ldhD-F/ldhD-R进行PCR验证,通过PCR,核酸电泳跑出来的胶图和设计引物时的预期是一致的转化子提供基因组送去生工生物测序,并将测序结果与源基因组比对,检验敲除结果,得到高光学纯L-乳酸工程菌。
进一步,还包括有步骤6:150μL甘油管保藏的拟干酪乳杆菌菌液,均匀涂布于茄子瓶斜面培养基中,在37℃条件下,持续36h,连续活化培养两代,用50mL无菌水将活化菌株从茄子瓶斜面培养基中洗脱下来,之后接入15%v/v接种量的种子摇瓶培养基中,摇床在37℃,100r/min的条件下振荡培养12h左右,当OD620=10后再接入20%v/v接种量的发酵摇瓶培养基中,摇床在37℃,110r/min条件下振荡培养48h。
进一步,还包括有步骤7:建立HPLC的方法对发酵产物中L-乳酸和D-乳酸对映体进行拆分,并检测发酵产物质中的比例,方法:采用手性柱phenomenx LC column色谱柱,流动相为0.002mol/L CuSO4·5H2O和5%异丙醇溶液,流速1mL/min,进样量5μL,检测波长254nm,柱温30℃,用化学纯的L-乳酸和D-乳酸作为标品制作标准曲线,然后将样品稀释到标准曲线的范围内进行检测。
利用所述方法制备得到的高光学纯L-乳酸工程菌也属于本发明的保护范围。
综上所述,本发明具有以下有益效果:
1、本发明选取拟干酪乳杆菌(Lactobacillus paracasei NCBIO01)作为出发菌株,参照其全基因组序列,基于CRISPR/Cas9基因编辑方法构建了pNcasA-△ldhD敲除质粒,无痕敲除D-乳酸脱氢酶(ldhD)的同时过表达一个L-乳酸脱氢酶基因(ldhL),得到一株高光学纯L-乳酸工程菌,使其L-乳酸光学纯度从原来的95.6%提高到99%以上,并且生产速率达到5.5g/L/h以上,成为潜在的工业L-乳酸高效生产菌株。
附图说明
图1(A)为sgRNA使用PCR产物通过核酸电泳检测结果;
图1(B)为上游同源臂、ldhL基因、氨苄抗性基因、下游同源臂分别使用PCR产物通过核酸电泳检测结果;
图1(C)为将sgRNA、上游同源臂、ldhL基因、氨苄抗性基因和下游同源臂融合PCR方式后通过核酸电泳检测结果;
图1(D)为大肠菌落使用PCR产物通过核酸电泳检测结果;
图2为完整敲除质粒示意图;
图3(A)为挑选13个转化子提取基因组,用ldhD内源引物验证结果;
图3(B)为ldhD外源引物验证结果;
图4为过表达菌株与出发菌株生长对比;
图5为过表达菌株和出发菌株L-乳酸生成耗糖对比;
图6为在5L罐中过表达菌株与出发菌株生长对比;
图7为过表达菌株和出发菌在5L发酵罐中的乳酸和葡萄糖变化;
图8为在5L罐中过表达菌株和出发菌株D-乳酸对比;
图9拟干酪乳杆菌出发菌株发酵生产L-乳酸过程中生长动力学拟合;
图10拟干酪乳杆菌过表达菌株发酵生产L-乳酸过程中生长动力学拟合;
图11为拟干酪乳杆菌感受态生长曲线。
具体实施方式
参照附图对本发明做进一步说明。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
如图1-图11所示,pNCas质粒及pMG36e质粒均由上海理工大学美国海洋光学联合实验室惠赠,pUC57-gRNA质粒由华大公司提供,大肠杆菌DH5α及感受态均由实验室保存和制备,37℃培养;拟干酪乳杆菌及感受态均由实验室保存,37℃培养。Cas9启动子来自乳酸菌强启动子,sgRNA启动子来自拟干酪乳杆菌L-乳酸脱氢酶启动子。LB培养基(g/L):胰蛋白胨10、酵母粉5、NaCl10,pH 7.0。固体培养基再加入2%的琼脂,用于大肠杆菌培养;MRS液体培养基(g/L):葡萄糖40,蛋白胨10,牛肉膏10,酵母膏10,柠檬酸二胺2,磷酸氢二钾2,硫酸镁0.2,硫酸锰0.2,氯化钠0.03,硫酸亚铁0.01,乙酸钠4,碳酸钙25,吐温-80 1mL,1mol/LNaOH溶液调pH至6.0;发酵培养基(g/L):葡萄糖90,蛋白胨13.33,酵母膏13.33,硫酸镁0.0133,硫酸锰0.0133,氯化钠0.0133,硫酸亚铁0.0133,乙酸钠0.67,碳酸钙40,1mol/LNaOH溶液调pH至6.0。MRS固体培养基:在上述MRS液体培养基中添加琼脂20g/L;MMRS培养基:上述MRS培养基80mL,0.5mol/L蔗糖,2mmol/L的CaCl2和20mmol/L的MaCl2加水补足至100mL。电转缓冲液:272mmol/L蔗糖,0.5mmol/L MgCl2,0.5mmol/L KH2PO4(pH 7.4)。以上培养基以及缓冲液均用去离子水配置。其中5L台式发酵罐上(工作体积为4L)进行分批发酵实验,初始葡萄糖浓度为130g/L,通气控制在0.02vvm,并用25%(w/w)氨水溶液作为中和剂全程调控发酵液pH在6.0,接种量为20%。
胶回收试剂盒(Gel Extraction Kit),质粒小提试剂盒(Plasmid Mini Kit),纯化试剂盒(Clear Up)均来自Axygen公司,DNA Maker、提基因组试剂盒、限制性内切酶、DNA连接酶均购自于Takara公司;一步克隆(One-Step Cloning Kit)连接试剂盒购自于南京诺唯赞公司。氯霉素(chloromycetin)和氨苄青霉素(ampicillin)以及红霉素(Erythromycin)均购于上海生工公司。
拟干酪乳杆菌D-乳酸脱氢酶(ldhD)基因的sgRNA由华大基因设计合成并连接到载体pUC57上。
一种高光学纯L-乳酸工程菌的构建方法,包括如下步骤,
步骤1:制备PCR体系50μl,取DNA模板1μl,在PCR扩增过程中,通过设计的引物对已知的DNA进行扩增来获得我们所需要的目的片段,所以就有这么一个DNA模板存在,PremixTaq Mi25μl,x是一个商业试剂,可直接从公司购买,上下游引物各1μl,扩增一个片段需要两条引物,正向/反向引物,这里的上下游引物也叫正向引物和反向引物,ddH2O 8.5μl,反应条件为95℃预变性5min,之后95℃变性30S,55℃退火30S,72℃延伸30S进行30个循环;最后72℃延伸6min,取PCR扩增产物,进行1%琼脂糖凝胶电泳鉴定,鉴定成功后对PCR产物进行纯化回收;
步骤2:根据拟干酪乳杆菌ldhD基因序列,在ldhD基因CDS区上游设计sgRNA,通过引物ldhDsg-F/ldhDsg-R PCR扩增得到sgRNA片段,以拟干酪乳杆菌基因组为模板,用引物ldhDup-F/ldhDup-R、ldhDdown-F/ldhDdown-R分别PCR扩增出ldhD上游同源臂和下游同源臂各1000bp,以ldhL-F/ldhL-R引物PCR扩增出L-乳酸脱氢酶基因(ldhL),以pan7-1质粒为模板,以Amp-F/Amp-R引物PCR扩增出氨苄青霉素抗性基因片段,
步骤3:再用PCR将氨苄青霉素抗性基因片段、上游同源臂、ldhL基因、下游同源臂以及sgRNA融合,引物设计时需要使融合的片段之间存在20-40bp的重复区,五个片段融合的摩尔比为1:3:5:3:1,得到构建完成的质粒pNcas-△ldhD;
步骤4:将保存好的Lactobacillus paracasei甘油管在一级MRS茄子瓶涂布活化,37℃培养36h。然后将一级MRS茄子瓶菌体转接到二级茄子瓶中,37℃静止培养18h。将二级茄子瓶中的菌体用50mL的无菌水洗涤,在MRS平板中划线,37℃静置培养36h,挑取单菌落接种至MRS液体培养基中,37℃,110r/min过夜培养,按照5%的比例转接至50mL含有1%浓度甘氨酸的MRS液体培养基中,37℃,110r/min培养至OD620值为0.6。然后4500rpm,4℃离心10min收集菌体,电转缓冲液洗两遍,用1mL预冷的电转缓冲液重悬菌体,按每管80μL分装于1.5mL离心管中,冻存于-80℃,取100ng的pNcas-△ldhD质粒,将其与感受态细胞充分混合均匀,转移至冰上预冷的2mm电极杯中,进行电转化,快速加入37℃预热的复苏液MMRS,转移至离心管中,37℃复苏培养3h,涂布抗性平板,37℃培养48h,计算其转化效率;
步骤5:用具有氨苄抗性的MRS平板筛选含有pNcas-△ldhD质粒的转化子,然后挑取转化子单菌落过夜复苏培养,提取突变株基因组用ldhD特异性引物ldhD-F/ldhD-R进行PCR验证,对验证成功的转化子提供基因组送去生工生物测序,并将测序结果与源基因组比对,检验敲除结果;
步骤6:150μL甘油管保藏的拟干酪乳杆菌菌液,均匀涂布于茄子瓶斜面培养基中,在37℃条件下,持续36h,连续活化培养两代,用50mL无菌水将活化菌株从茄子瓶斜面培养基中洗脱下来,之后接入(按15%v/v接种量)种子摇瓶培养基中,摇床(37℃,100r/min)振荡培养12h左右(OD620=10)后再接入(按20%v/v接种量)发酵摇瓶培养基中,摇床(37℃,110r/min)振荡培养48h;
步骤7:建立HPLC的方法对发酵产物中L-乳酸和D-乳酸对映体进行拆分,并检测发酵产物质中的比例,方法:采用手性柱phenomenx LC column色谱柱。流动相为0.002mol/LCuSO4·5H2O和5%异丙醇溶液,流速1mL/min,进样量5μL,检测波长254nm,柱温30℃,用化学纯的L-乳酸和D-乳酸作为标品制作标准曲线,然后将样品稀释到标准曲线的范围内进行检测。
步骤5中的验证成功,举例说明,如用ldhD-F/ldhD-R这对引物进行PCR扩增,如果敲除成功的转化子扩增出来的电泳条带为1300bp,没有敲除成功的转化子则是1000bp
测试实验1:经过全基因组比对后发现拟干酪乳杆菌在NCBIO中只注释含有一个ldhD基因,两个L-乳酸脱氢酶(ldhL1和ldhL2),根据数据库比对后,我们将ldhL1作为过表达基因替换ldhD,步骤2和步骤3中,PCR获得的sgRNA的结果用3%的琼脂凝胶电泳检测结果如图1(A)所示,条带大小符合预期。氨苄抗性基因、过表达ldhL1基因、上游同源臂和下游同源臂PCR结果用1%的琼脂凝胶电泳检测结果如图1(B)所示,目的条带符合预期,用验证引物JP-F/JP-R进行大肠杆菌菌落PCR验证如图1(D)所示。构建后的完整敲除质粒pNcas-△ldhD如图2所示。融合PCR获得的产物如图1(C)所示。
表1 本研究使用引物
上述使用引物均由生工生物工程(上海)股份有限公司合成。
测试实验2:将构建好的质粒pNcas-△ldhD通过电转化到拟干酪乳杆菌(Lactobacillus paracasei)感受态中,涂布在含有氨苄抗性的MRS平板上37℃培养48h,挑取长势较好的单菌落在平板上二次划线再次培养36h,然后挑取长势好的13株单菌落接入试管中过夜培养后提取基因组,在靶基因内设计一对内源验证引物YZ-F/YZ-R进行PCR检测这几株菌的敲除情况,结果如图3(A)发现有两株无目的条带,又在靶基因外侧上下游设计了一对外源验证引物ldhDZ-F/ldhDZ-R对两株突变株进行PCR,并以原始菌株作对照,如图3(B)所示,若过表达ldhL基因成功替换被敲除ldhD基因,电泳条带应是1300bp,符合预期。进一步测序检验证明这两株突变株改造成功。
测试实验3:将过表达菌株和出发菌株在初始葡萄糖为90g/L的摇瓶中发酵培养,发酵结束后用SBA-40C生物传感分析仪测定发酵液中残余葡萄糖,并用HPLC的方法检测发酵液中L/D乳酸的含量(表2)。分别从细胞生长(图4)和乳酸生成以及葡萄糖消耗随时间的变化(图5)进行分析。
表2 过表达型菌株和出发型菌株乳酸发酵特性分析
将过表达型拟干酪乳杆菌与野生型菌株在37℃,110rpm条件下进行乳酸发酵过程特性的比较发现,在乳酸发酵过程中过表达型菌株的生长速率和乳酸的生产速率都比野生型菌株高,细胞量OD620从原来的20.25提高到23.51。发酵液最终pH基本维持一致,维持在5.8左右。过表达突变株发酵L-乳酸光学纯度达99%,与出发菌株的差异显著。经计算,缺失突变体菌株L-乳酸产量为82.9g/L,出发菌L-乳酸产量为71g/L。
测试实验3:
过表达菌株和出发菌株在5L罐上进行发酵实验,并从菌体的生长、L/D-乳酸生产、葡萄糖消耗过程进行分析。如图6,与出发菌株相比过表达菌株的菌浓提高了10.6%,并通过Logistic方程和L-P方程拟合两株菌菌体生长的过程(如图9、10),发现过表达菌株μmax达到0.497h-1,相比出发菌株提高了12.3%。从图7中可以看出,整个乳酸发酵周期为22h,发酵结束时发酵液中的葡萄糖完全耗尽,过表达菌株L-乳酸最终达到121.6g/L,出发菌株L-乳酸达到112.8g/L。过表达菌株相比出发菌株具有更高L-乳酸的生产速率,达到5.52g/L/h,提高了7.6%,和已有报道的凝结芽孢杆菌ATCC7050相比,经过改造后的凝结芽孢杆菌ATCC7050产酸速率为1.79g/L/h,拟干酪乳杆菌过表达菌株产酸速率要高出其2倍。在葡萄糖消耗量相同的情况下,过表达菌株具有更高的L-乳酸转化率,达到94.5%,比出发菌89.6%的L-乳酸转化率提高了4.9%。图8过表达菌株和出发菌株D-乳酸含量随时间变化可发现,当ldhD基因敲除后,D-乳酸含量相对出发菌株有了大幅度减少,使得L-乳酸的光学纯度可达到99%。
经分析,敲除ldhD基因后,阻断了D-乳酸代谢途径,更多的前体物质流向L-乳酸代谢途径。此外,突变菌株的乳酸累计不受ldhD基因缺失的影响,证明了ldhD基因不是拟干酪乳杆菌乳酸代谢中乳酸产量的限制性因素。ldhL基因的过表达使拟干酪乳杆菌的生长速率加快,并可以维持更高的细胞量,乳酸的生产速率提高,缩短了乳酸发酵的周期。
乳酸菌中的表达质粒pMG36e作为对照质粒,检验不制备的电转感受态细胞的转化效率。
拟干酪乳杆菌电转条件的优化
不同宿主在电击转化条件方面都有差异,都需要根据宿主自身来做调整和优化。在拟干酪乳杆菌中,影响转化的因素主要有收集感受态细胞时的菌体浓度、电转缓冲液的选择、在培养过程中加入甘氨酸的的浓度以及在电击过程中电压大小的控制。拟干酪乳杆菌感受态制作方法如第二章,在制作感受态过程中,细胞的生长时期尤为重要,一般取对数生长时期的感受态细胞来收集菌体制作感受态。将拟干酪乳杆菌单菌落接种至20ml的MRS培养基中过夜培养,再取出15ml接种至100ml含有MRS液体培养基的摇瓶中培养,每隔1h取出1ml样品检测OD620值,以吸光度为纵坐标,时间为横坐标,绘制感受态的生长曲线如图11所示,可看出拟干酪乳杆菌在3h后进入对数生长期,6h后进入平台期。
根据拟干酪乳杆菌感受态生长曲线选择前、中、后三个不同时期的细胞制作感受态(细胞的OD620分别为0.3、0.6、0.9),在电压为1.0kv,甘氨酸浓度为1%,电转缓冲液为10%甘油的相同情况下得到其转化效率如表3.1所示。
表3.1 干酪乳杆菌菌体生长状态对电转化效率影响
由表3.1和图11可清晰看出在其余条件相同的情况下,OD620在0.6时转化子数量最多,因此选择下OD620=0.6时收集感受态细胞为最优条件。
随后对电转缓冲液进行探索,根据已有的文献报道分别选择了HEB缓冲液,10%甘油,PEB-1缓冲液作为研究对象,在OD620=0.6时收集菌体,电压为1.0kv,甘氨酸浓度为1%。
表3.2 缓冲液对电转化效率影响
显然由表可知,当在其他参数条件一致的情况下,使用PEB-1缓冲液相比其他缓冲液有更高的电转效率。
在确定了最适的菌浓和电转缓冲液后,进一步对甘氨酸浓度进行探究。由于甘氨酸可以起到弱化细胞壁的作用,从而增加细胞的通透性,有利于电转过程中将外源DNA导入到受体细胞内,因此在制作乳酸菌感受态过程中都会在MRS液体培养基中加入合适比例的甘氨酸。在上述研究的基础上,选择PEB-1缓冲液,在OD620=0.6时收集菌体,电压为1.0kv,甘氨酸浓度分别为0.5%,1%,2%进行探究,结果如表3.3所示。
表3.3 甘氨酸浓度对电转化效率影响
实验结果表明,当甘氨酸浓度为1%时,由最高的转化效率。
最后对电击转化过程中电压进行探究,合适的电压尤为关键,它使细胞处于一种最适接受外源DNA的临界状态,弱电压过高会导致原本脆弱的的细胞死亡,若电压过低会使外源DNA无法导入细胞。因此在确定了以上的条件下对电转化的电压参数进行探究,择PEB-1缓冲液,在OD620=0.6时收集菌体,1%的甘氨酸浓度,分别在电压为0.8kv,1.5kv,2.0kv,2.5kv条件下进行探究,结果如表3.4所示。
表3.4 电压对电转化效率影响
由实验数据统计的当电压在1.5kv时有最高的电转效率,往后如果增加电压反而会降低转化效率。
通过对拟干酪乳杆菌整个转化过程的优化得出了一个最优的电转条件。总结:最适收集菌体是在OD620=0.6时,电转缓冲液选择PEB-1缓冲液,菌体培养过程中加入1%浓度甘氨酸,最适的电压条件为1.5kv。
按照以上条件进行电转化,拟干酪乳杆菌的电转效率1.25×103CFU/μg。
以上所述仅为本发明的较佳实施例,并不用于限制本发明,凡在本发明的设计构思之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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<110> 华东理工大学
华东理工大学青岛创新研究院
<120> 一种高光学纯L-乳酸工程菌的构建方法
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Claims (3)
1.一种高光学纯L-乳酸工程菌的构建方法,其特征在于:所述构件方法包括:
步骤1:制备PCR体系50μl,取PCR扩增产物,进行1%琼脂糖凝胶电泳鉴定,鉴定成功后对PCR产物进行纯化回收;
步骤2:根据拟干酪乳杆菌ldhD基因序列,在ldhD基因CDS区上游设计sgRNA,通过引物ldhDsg-F/ldhDsg-R PCR扩增得到sgRNA片段,以拟干酪乳杆菌基因组为模板,用引物ldhDup-F/ldhDup-R、ldhDdown-F/ldhDdown-R分别PCR扩增出ldhD上游同源臂和下游同源臂各1000bp,以ldhL-F/ldhL-R引物PCR扩增出L-乳酸脱氢酶基因(ldhL),以pan7-1质粒为模板,以Amp-F/Amp-R引物PCR扩增出氨苄青霉素抗性基因片段;
步骤3:再用PCR将氨苄青霉素抗性基因片段、上游同源臂、ldhL基因、下游同源臂以及sgRNA融合,引物设计时需要使融合的片段之间存在20-40bp的重复区,五个片段融合的摩尔比为1:3:5:3:1,得到构建完成的质粒pNcas-△ldhD;
步骤4:将保存好的Lactobacillus paracasei甘油管在一级MRS茄子瓶涂布活化,37℃培养36h,然后将一级MRS茄子瓶菌体转接到二级茄子瓶中,37℃静止培养18h,将二级茄子瓶中的菌体用50mL的无菌水洗涤,在MRS平板中划线,37℃静置培养36h,挑取单菌落接种至MRS液体培养基中,37℃,110r/min过夜培养,按照5%的比例转接至50mL含有1%浓度甘氨酸的MRS液体培养基中,37℃,110r/min培养至OD620值为0.6,然后4500rpm,4℃离心10min收集菌体,电转缓冲液洗两遍,用1mL预冷的电转缓冲液重悬菌体,按每管80μL分装于1.5mL离心管中,冻存于-80℃,取100ng的pNcas-△ldhD质粒,将其与感受态细胞充分混合均匀,转移至冰上预冷的2mm电极杯中,进行电转化,快速加入37℃预热的复苏液MMRS,转移至离心管中,37℃复苏培养3h,涂布抗性平板,37℃培养48h,计算其转化效率;
步骤5:用具有氨苄抗性的MRS平板筛选含有pNcas-△ldhD质粒的转化子,然后挑取转化子单菌落过夜复苏培养,提取突变株基因组用ldhD特异性引物ldhD-F/ldhD-R进行PCR验证,通过PCR,核酸电泳跑出来的胶图和设计引物时的预期是一致的的转化子提供基因组送去生工生物测序,并将测序结果与源基因组比对,检验敲除结果,得到高光学纯L-乳酸工程菌。
2.根据权利要求1所述的高光学纯L-乳酸工程菌的构建方法,其特征在于:还包括有步骤6:150μL甘油管保藏的拟干酪乳杆菌菌液,均匀涂布于茄子瓶斜面培养基中,在37℃条件下,持续36h,连续活化培养两代,用50mL无菌水将活化菌株从茄子瓶斜面培养基中洗脱下来,之后接入15%v/v接种量的种子摇瓶培养基中,摇床在37℃,100r/min的条件下振荡培养12h左右,当OD620=10后再接入20%v/v接种量的发酵摇瓶培养基中,摇床在37℃,110r/min的条件下振荡培养48h。
3.根据权利要求2所述的高光学纯L-乳酸工程菌的构建方法,其特征在于:还包括有步骤7:建立HPLC的方法对发酵产物中L-乳酸和D-乳酸对映体进行拆分,并检测发酵产物质中的比例,方法:采用手性柱phenomenx LC column色谱柱,流动相为0.002mol/L CuSO4·5H2O和5%异丙醇溶液,流速1mL/min,进样量5μL,检测波长254nm,柱温30℃,用化学纯的L-乳酸和D-乳酸作为标品制作标准曲线,然后将样品稀释到标准曲线的范围内进行检测。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114875045A (zh) * | 2022-04-21 | 2022-08-09 | 广西工业职业技术学院 | 一种高产l-乳酸的鼠李糖乳杆菌基因工程菌的构建方法 |
CN114875079A (zh) * | 2022-04-21 | 2022-08-09 | 广西工业职业技术学院 | 一种产高光学纯l-乳酸的鼠李糖乳杆菌基因工程菌的构建方法 |
CN115011537A (zh) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | 一株双厌氧启动子诱导产高光学纯l-乳酸的工程菌及其制备方法与应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013162274A1 (ko) * | 2012-04-24 | 2013-10-31 | 씨제이제일제당 (주) | 신규한 d형 젖산 생산균주 및 그의 용도 |
CN109321590A (zh) * | 2018-10-19 | 2019-02-12 | 北京化工大学 | 利用乙酸生产l-乳酸的基因工程菌及其构建方法和应用 |
CN111500517A (zh) * | 2020-05-27 | 2020-08-07 | 吉林中粮生化有限公司 | 产l-乳酸的重组菌株及其构建方法和发酵产l-乳酸的方法及应用 |
-
2020
- 2020-09-08 CN CN202010936756.2A patent/CN111849852A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013162274A1 (ko) * | 2012-04-24 | 2013-10-31 | 씨제이제일제당 (주) | 신규한 d형 젖산 생산균주 및 그의 용도 |
CN109321590A (zh) * | 2018-10-19 | 2019-02-12 | 北京化工大学 | 利用乙酸生产l-乳酸的基因工程菌及其构建方法和应用 |
CN111500517A (zh) * | 2020-05-27 | 2020-08-07 | 吉林中粮生化有限公司 | 产l-乳酸的重组菌株及其构建方法和发酵产l-乳酸的方法及应用 |
Non-Patent Citations (6)
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114875045A (zh) * | 2022-04-21 | 2022-08-09 | 广西工业职业技术学院 | 一种高产l-乳酸的鼠李糖乳杆菌基因工程菌的构建方法 |
CN114875079A (zh) * | 2022-04-21 | 2022-08-09 | 广西工业职业技术学院 | 一种产高光学纯l-乳酸的鼠李糖乳杆菌基因工程菌的构建方法 |
CN115011537A (zh) * | 2022-06-14 | 2022-09-06 | 湖北工业大学 | 一株双厌氧启动子诱导产高光学纯l-乳酸的工程菌及其制备方法与应用 |
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