CN113462630B - 一种高产d-泛酸基因工程菌及其构建与应用 - Google Patents
一种高产d-泛酸基因工程菌及其构建与应用 Download PDFInfo
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Abstract
本发明涉及基因工程领域,具体涉及一种高产泛酸基因工程菌及其构建方法,以及其在微生物发酵制备泛酸中的应用。本发明通过突变其基因组中panB基因的关键位点来加强panB基因的表达,有利于提高生物发酵法生产泛酸的效率。本发明得到了一系列对泛酸产率有明显改变的酮泛解酸羟甲基转移酶突变基因工程菌,所得酮泛解酸羟甲基转移酶突变基因工程菌用于发酵法生产D‑泛酸,相较原始基因工程菌株,本发明构建的酮泛解酸羟甲基转移酶突变基因工程菌通过发酵生产D‑泛酸的产率有所提高,具有工业化开发应用前景。
Description
(一)技术领域
本发明属于生物技术领域,具体涉及一种高产D-泛酸基因工程菌及其构建方法,以及其在微生物发酵制备D-泛酸中的应用。
(二)背景技术
D-泛酸,又称遍多酸或维生素B5,是一种水溶性维生素,D-泛酸的化学索引号(CAS)为79-83-4,相对分子质量为219.23500,相对密度为1.266g/cm3,其化学式为CH2OHC(CH3)2CHOHCONHCH2CH2COOH,由泛解酸和β-Ala组成。有旋光性,仅D型([α]=+37.5°)有生物活性。消旋泛酸具有吸湿性和静电吸附性;纯游离泛酸是一种淡黄色粘稠的油状物,具酸性,易溶于水和乙醇,不溶于苯和氯仿,泛酸在酸、碱、光及热等条件下都不稳定。泛酸是脂肪酸合成类固醇所必需的物质;也可参与类固醇紫质、褪黑激素和亚铁血红素的合成;还是体内柠檬酸循环、胆碱乙酰化、合成抗体等代谢所必需的中间物。泛酸在体内可作用于正常的上皮器官如神经、肾上腺、消化道及皮肤,提高动物对病原体的抵抗力。泛酸也可以增加谷胱甘肽的生物合成从而减缓细胞凋亡和损伤。泛酸及其衍生物还可以减轻抗生素等药物引起的毒副作用,参与多种营养成分的吸收和利用。
目前泛酸的主要生产方式有以下几种:(1)物理诱导结晶法:利用混旋泛酸钙的溶解度大于D-型或L-型这一特点进行诱导结晶,此方法工艺成熟,但只能生产泛酸钙,无法用于其他泛酸衍生物的生产。(2)化学法:先通过化学法制备得到β-丙氨酸和DL-泛解酸内酯两种关键中间体再用于合成D-泛酸。其中β-丙氨酸主要通过丙烯腈法、丙烯酸法和琥珀酰亚胺法合成得到,而DL-泛解酸内酯主要通过异丁醛-甲醛法、异丁醛-醛乙酸法和异丁醛-三氯甲烷法得到。(3)生物酶法:泛酸可以通过泛酸合成酶(pantothenate synthase,panC)催化泛酮与β-丙氨酸发生ATP依赖的缩合反应合成。(4)微生物发酵法:通过代谢及基因工程手段对泛酸生物途径(泛酸代谢途径图参见图1)的关键基因进行改造,得到高产D-泛酸的菌株。
自上个世纪90年代开始,一些著名的化学制药公司BASF、DSM和Degussa AG等开始关注D-泛酸发酵法合成。Miki Hiroshi等人通过运用紫外诱变和亚硝基胍诱变技术与特殊培养基筛选相结合进行高产D-泛解酸菌株的选育,筛选得到一株高产D-泛酸的基因工程菌,该菌株需添加外源β-丙氨酸,经过72h补料发酵后D-泛酸产量可达65.4g/L(Processfor producing D-pantoic acid and D-pantothenicacid or salts thereof:US5932457[P].1999-08-03.)。Rogers R.Yocum等人致力于构建高产D-泛酸的枯草芽孢杆菌菌株(Bacillus subtilis),通过解除panBCD、ilvBNC、panE和ilvD的调控来增强D-泛酸合成,构建过表达panBD的质粒,能有效促进D-泛酸积累(Microorganisms and processesfor enhancedproduction of pantothenate.US 7291489[P].2007-11-06.)。RubioAileen等人发现通过过表达panB(Salmonella enterica serovar Typhimurium)可以提高D-泛酸的产量(Elevated levels of ketopantoate hydroxymethyltransferase(PanB)lead to a physiologically significant coenzyme A elevation in Salmonellaenterica serovar Typhimurium.[J].Journal of bacteriology,2002,184(10))。
(三)发明内容
为了提高生物发酵法合成D-泛酸的产率,降低底物反馈抑制,本发明提供一种高产D-泛酸基因工程菌及其构建方法,以及其在微生物发酵制备D-泛酸中的应用。
本发明采用的技术方案是:
一种高产D-泛酸基因工程菌,以大肠杆菌DPA-11-pTrc99A为底盘菌,突变其基因组中panB基因的关键位点,将SEQ ID NO:1所示氨基酸序列的18位、20位、21位、24位、25位、26位、27位、47位、48位、59位、89位和185位进行单突变;或者将SEQ ID NO:1所示氨基酸序列第21与24位、第24与25位、第24与27位、第59与61位进行双位点突变;或者将SEQ ID NO:1所示氨基酸序列第21、26、27位或者第25、26、27进行三位点突变获得。
优选的,所述单点突变为下列之一:第18位谷氨酸突变为丙氨酸;第20位赖氨酸突变为丙氨酸;第21位缬氨酸突变为谷氨酸;第24位谷氨酰胺突变为丙氨酸;第25位的赖氨酸突变为丙氨酸;第26位缬氨酸突变为天冬酰胺;第27位丝氨酸突变为丙氨酸;第47位蛋氨酸突变为异亮氨酸;第48位亮氨酸突变为异亮氨酸;第59位亮氨酸突变为苯基丙氨酸;第89位缬氨酸突变为异亮氨酸;第185位谷丙酸突变为丝氨酸(氨基酸序列为SEQ ID NO:3,编码基因序列为SEQ ID NO:4)。
所述双位点突变为下列之一:(1)第21位缬氨酸突变为谷氨酸,24位谷氨酸突变为丙氨酸;(2)第24位谷氨酰胺突变为丙氨酸,第25位的赖氨酸突变为丙氨酸;(3)第24位谷氨酰胺突变为丙氨酸,第27位丝氨酸突变为丙氨酸;(4)第59位的亮氨酸突变为苯基丙氨酸,第61位的精氨酸突变为苯基丙氨酸。
所述三位点突变为下列之一:(1)第21位缬氨酸突变为谷氨酸,第26位缬氨酸突变为天冬酰胺,第27位丝氨酸突变为丙氨酸;(2)第25位赖氨酸突变为丙氨酸,第26位缬氨酸突变为天冬酰胺,第27位丝氨酸突变为丙氨酸。
本发明通过突变其基因组中panB基因的关键位点来提高panB基因的活力,有利于提高生物发酵法生产D-泛酸的效率。
具体的,所述突变体是将SEQ ID NO:1所示氨基酸(编码基因序列为SEQ ID NO:2)序列经下列之一的突变获得:(1)第18位谷氨酸突变为丙氨酸(E18A);(2)第20位的赖氨酸突变为丙氨酸(K20A);(3)第21位缬氨酸突变为谷氨酸(V21E);(4)第24位谷氨酰胺突变为丙氨酸(Q24A);(5)第25位的赖氨酸突变为丙氨酸(K25A);(6)第26位缬氨酸突变为天冬酰胺(V26N);(7)第27位丝氨酸突变为丙氨酸(S27A);(8)第47位蛋氨酸突变为异亮氨酸(M47I);(9)第48位亮氨酸突变为异亮氨酸(L48I);(10)第59位亮氨酸突变为苯基丙氨酸(L59F);(11)第89位缬氨酸突变为异亮氨酸(V89I);(12)第185位丙氨酸突变为丝氨酸(A185S);(13)第21位缬氨酸突变为谷氨酸与第24位谷氨酰胺突变为丙氨酸(V21E/Q24A);(14)第24位谷氨酰胺突变为丙氨酸与第25位的赖氨酸突变为丙氨酸(Q24A/K25A);(14)第24位谷氨酰胺突变为丙氨酸与第27位丝氨酸突变为丙氨酸(Q24A/S27A);(16)第59位的亮氨酸突变为苯基丙氨酸与第61位的精氨酸突变为苯基丙氨酸(L59F/R61F);(17)第21位缬氨酸突变为谷氨酸,第24位谷氨酰胺突变为丙氨酸与第27位丝氨酸突变为丙氨酸(V21E/Q24A/S27A);(18)第25位赖氨酸突变为丙氨酸,第26位缬氨酸突变为天冬酰胺,第27位丝氨酸突变为丙氨酸(K25A/V26N/S27A)。
本发明所述突变后的基因工程菌株是以菌株DPA-11-pTrc99A-panBpanC号工程菌为底盘菌株,突变其基因组中panB基因的关键位点,过表达panB,进而提高泛酸的产率。首先设计相关突变位点的上下游引物,通过PCR技术进行定点突变,然后将消化好的PCR产物转化至E.coli DH5α(克隆宿主)中,测序结果正确后,将构建好的突变体质粒导入菌株DPA-11-pTrc99A中,得到突变后的基因工程菌株。对所得基因工程菌进行发酵培养,对所得发酵上清液进行HPLC检测得到D-泛酸产率明显改变的突变体。
本发明还涉及构建所述高产D-泛酸基因工程菌的方法,所述方法包括:大肠杆菌DPA-11-pTrc99A-panBpanC为底盘菌,突变其基因组中panB基因的关键位点,进行单点突变,将SEQ ID NO:1所示氨基酸序列的第18位谷氨酸突变为丙氨酸、第20位赖氨酸突变为丙氨酸、第21位缬氨酸突变为谷氨酸、第24位谷氨酰胺突变为丙氨酸、第25位的赖氨酸突变为丙氨酸、第26位缬氨酸突变为天冬酰胺、第27位丝氨酸突变为丙氨酸、第47位蛋氨酸突变为异亮氨酸、第48位亮氨酸突变为异亮氨酸、第59位亮氨酸突变为苯基丙氨酸、第89位缬氨酸突变为异亮氨酸、第185位谷丙酸突变为丝氨酸;或者进行双位点突变,将SEQ ID NO:1所示氨基酸序列第21位缬氨酸突变为谷氨酸、24位谷氨酸突变为丙氨酸,第24位谷氨酰胺突变为丙氨酸、第25位的赖氨酸突变为丙氨酸,第24位谷氨酰胺突变为丙氨酸、第27位丝氨酸突变为丙氨酸,第59位的亮氨酸突变为苯基丙氨酸、第61位的精氨酸突变为苯基丙氨酸;或者进行三位点突变,将SEQ ID NO:1所示氨基酸序列第21位缬氨酸突变为谷氨酸、第26位缬氨酸突变为天冬酰胺、第27位丝氨酸突变为丙氨酸,或者第25位赖氨酸突变为丙氨酸、第26位缬氨酸突变为天冬酰胺、第27位丝氨酸突变为丙氨酸,获得所述高产D-泛酸基因工程菌。
所述大肠杆菌DPA-11-pTrc99A-panBpanC按如下方法构建获得:
(1)应用CRISPR-Cas9基因编辑技术将底盘菌E.coli W3110基因组中的panC、panB、panE和ilvC基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC;
(2)应用CRISPR-Cas9基因编辑技术在菌株E.coli W3110 Trc-panCpanEpanBilvC基因组中修复ilvG基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*;
(3)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*中avtA基因敲除,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA;
(4)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA上ilvE基因的起始密码子ATG替换为GTG,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*;
(5)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*中coaA编码的酶第106位氨基酸由丙氨酸突变为精氨酸,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*;
(6)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*中ilvA基因敲除,得到菌株E.coli W3110Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA;
(7)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA中lpd基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd;
(8)应用CRISPR-Cas9基因编辑技术敲除菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd中的glk基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk;
(9)以质粒pTrc99A(pTrc99A质粒由Addgene公司购买获得,产品编号为VT1294)为模板,通过PCR扩增获取线性化质粒片段,同样采用PCR扩增技术分别获得panB及panC目的片段,再采用诺唯赞公司的一步克隆试剂盒将质粒框架和目的片段无缝连接,连接好的产物及时转化到E.coli DH5α感受态中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,获得pTrc99A-panBpanC菌株;
(10)将构建好的pTrc99A-panBpanC菌株的质粒导入步骤(8)构建的W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk感受态细胞中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,即为所述大肠杆菌DPA-11-pTrc99A-panBpanC。
本发明还涉及所述高产D-泛酸基因工程菌在微生物发酵制备D-泛酸中的应用。
具体的,所述应用为:将所述高产D-泛酸基因工程菌接种至发酵培养基,进行摇瓶发酵制备D-泛酸。
通常,所述基因工程菌发酵前,先接种至LB培养基中,于温度37℃,转速180rpm的摇床上过夜培养,然后以体积浓度5%接种量接种到发酵培养基中培养。所述发酵培养基M-1的配方如下:葡萄糖20g/L,硫酸铵16g/L,酵母粉2g/L,KH2PO4 0.8g/L,MgSO4 0.5g/L,CaCO3 15g/L,盐溶液(其中包括CuCl2 10g/L,FeSO4·7H2O 10g/L,ZnSO4·H2O 1g/L,CuSO40.2g/L,NiCl2·7H2O 0.02g/L),接种时加入0.3g CaCO3,IPTG(终浓度为0.1mM)和Amp抗性(终浓度50mg/L),L-异亮氨酸(终浓度20mg/L),VB1(终浓度5mg/L),VB12(终浓度2mg/L)。(注:D-Pa-11为L-异亮氨酸缺陷型,则需在摇培养时添L-异亮氨酸(终浓度20mg/L)。发酵培养温度为30℃,发酵时间为48h。
本发明的有益效果主要体现在:本发明得到了一系列对D-泛酸产率有明显改变的酮泛解酸羟甲基转移酶突变基因工程菌,所得酮泛解酸羟甲基转移酶突变基因工程菌用于发酵法生产D-泛酸,相较原始基因工程菌株DPA-11-pTrc99A,本发明构建的酮泛解酸羟甲基转移酶突变基因工程菌DPA-11-panB-E18A,DPA-11-panB-K20A,DPA-11-panB-V21E,DPA-11-panB-K25A,DPA-11-panB-V26N,DPA-11-panB-Q24A,DPA-11-panB-S27A,DPA-11-panB-M47I,DPA-11-panB-L48I,DPA-11-panB-L59F,DPA-11-panB-V89I,DPA-11-panB-A185S,DPA-11-panB-V21E/Q24A,DPA-11-panB-Q24A/K25A,DPA-11-panB-Q24A/S27A,DPA-11-panB-L59F/R61F,DPA-11-panB-V21E/V26N/S27A,DPA-11-panB-K25A/V26N/S27A通过发酵生产D-泛酸的产率有所提高,具有工业化开发应用前景。
(四)附图说明
图1为D-泛酸代谢途径图。
图2为实施例中3中对panB基因上的关键位点通过PCR扩增技术突变后的琼脂糖凝胶电泳图。其中M:DNA分子量marker(250bp),泳道2-10分别为E18A,K20A,V21E,Q24A,K25A,V26N,S27A,M47I,泳道12-14分别为L48I,L59F,V89I,A185S。
图3为实施例中4,5中对panB基因上的关键位点通过PCR扩增技术突变后的琼脂糖凝胶电泳图。其中M:DNA分子量marker(250bp),泳道2为V21E/Q24A,泳道4-8分别为,Q24A/K25A,Q24A/S27A,L59F/R61F,V21E/V26N/S27A,K25A/V26N/S27A。
图4为实施例中8中的D-泛酸浓度标准曲线图。
图5为实施例中9中的产物D-泛酸的HPLC液相谱图。
图6位实施例中9和10中的D-泛酸产量和OD600柱形图。
(五)具体实施方式
下面结合具体实例对本发明做进一步详细地说明,但本发明并不限于以下实施例:
本发明中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例1:制备E.coli DH5α感受态
将-80℃保藏的E.coli DH5α于无抗LB平板划线,37℃培养12h,长出单菌落。挑取单菌落接种于10mL LB液体培养基,然后于37℃,150rpm振荡培养12h。以1%的接种量转接至50mL LB液体培养基,然后于37℃,150rpm振荡培养2h左右,当培养物OD600接近0.6-0.8时冰浴30min以上使其停止生长。后在4℃条件下,5000rpm离心5min收集细胞,弃上清。后加入20mL预冷的0.1M CaCl2溶液重悬,冰浴30min。重复离心和加入0.1M CaCl2溶液重悬操作。4℃,5000rpm离心10min收集细胞,弃上清。加入1mL(每50mL培养基)含15%甘油的0.1MCaCl2溶液重悬细胞,分装100μL/管,并于-80℃保藏备用。
实施例2:制备菌株DPA-11感受态
pTrc99A质粒由Addgene公司购买获得,产品编号为VT1294。菌株DPA-11和菌株DPA-11-pTrc99A-panBpanC为浙江工业大学生物工程学院实验室构建与保藏。
菌株DPA-11构建:
(1)应用CRISPR-Cas9基因编辑技术将底盘菌E.coli W3110基因组中的panC、panB、panE和ilvC基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC;
(2)应用CRISPR-Cas9基因编辑技术在菌株E.coli W3110 Trc-panCpanEpanBilvC基因组中修复ilvG基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*;
(3)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*中avtA基因敲除,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA;
(4)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA上ilvE基因的起始密码子ATG替换为GTG,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*;
(5)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*中coaA编码的酶第106位氨基酸由丙氨酸突变为精氨酸,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*;
(6)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*中ilvA基因敲除,得到菌株E.coli W3110Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA;
(7)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA中lpd基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd;
(8)应用CRISPR-Cas9基因编辑技术敲除菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd中的glk基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk,记为DPA-11。
菌株DPA-11-pTrc99A-panBpanC的构建:
(1)pTrc99A质粒由Addgene公司购买获得,产品编号为VT1294。。
(2)首先以pTrc99A为模板,通过PCR扩增获取线性化质粒片段,同样采用PCR扩增技术分别获得panB及panC目的片段,再采用诺唯赞公司的一步克隆试剂盒将质粒框架和目的片段无缝连接,连接好的产物及时转化到E.coli DH5α感受态中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,获得pTrc99A-panBpanC菌株;
(3)将构建好的pTrc99A-panBpanC菌株的质粒导入基因编辑构建的W3110Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk感受态细胞中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,即为所述大肠杆菌DPA-11-pTrc99A-panBpanC。
菌株DPA-11感受态的制备:
将-80℃保藏的菌株DPA-11于无抗LB平板划线,37℃培养12h,长出单菌落。挑取单菌落接种于10mL LB液体培养基,然后于37℃,150rpm振荡培养12h。以1%的接种量转接至50mL LB液体培养基,然后于37℃,150rpm振荡培养2h左右,当培养物OD600接近0.6-0.8时冰浴30min以上使其停止生长。后在4℃条件下,5000rpm离心5min收集细胞,弃上清。后加入20mL预冷的0.1M CaCl2溶液重悬,冰浴30min。重复离心和加入0.1M CaCl2溶液重悬操作。4℃,5000rpm离心10min收集细胞,弃上清。加入1mL(每50mL培养基)含15%甘油的0.1MCaCl2溶液重悬细胞,分装100μL/管,并于-80℃保藏备用。
实施例3:构建单突变位点文库
通过Swiss-Model对谷氨酸棒杆菌来源的panB进行3D模型的构建,并使用AutoDock分子对接软件将KPHMT(panB)的模型和底物小分子α-酮异戊酸和5,10-亚甲基四氢叶酸进行分子对接,选取panB基因上的关键位点进行突变。设计引物构建单突变位点基因工程菌。
设计引物如下:
E18A
上游引物1:ACCCGTCATTTCCGCGCTGCTAAAGTAAACGGC
下游引物2:AGCGCGGAAATGACGGGTGCGGATTTTCTTTGC
K20A
上游引物3:CATTTCCGCGAAGCTGCTGTAAACGGCCAGAAA
下游引物4:AGCAGCTTCGCGGAAATGACGGGTGCGGATTTT
V21E
上游引物5:TTCCGCGAAGCTAAAGAGAACGGCCAGAAAGTT
下游引物6:CTCTTTAGCTTCGCGGAAATGACGGGTGCGGAT
Q24A
上游引物7:GCTAAAGTAAACGGCGCTAAAGTTTCGGTTCTC
下游引物8:AGCGCCGTTTACTTTAGCTTCGCGGAAATGACG
K25A
上游引物9:AAAGTAAACGGCCAGGCTGTTTCGGTTCTCACC
下游引物10:AGCCTGGCCGTTTACTTTAGCTTCGCGGAAATG
V26N
上游引物11:GTAAACGGCCAGAAAACCTCGGTTCTCACCAGC
下游引物12:GGTTTTCTGGCCGTTTACTTTAGCTTCGCGGAA
S27A
上游引物13:AACGGCCAGAAAGTTGCTGTTCTCACCAGCTAT
下游引物14:AGCAACTTTCTGGCCGTTTACTTTAGCTTCGCG
M47I
上游引物15:GAGGCTGGCGTCGATATCCTCCTTGTTGGTGAT
下游引物16:GATATCGACGCCAGCCTCATCAAAAATGCGCGC
L48I
上游引物17:GCTGGCGTCGATATGATCCTTGTTGGTGATTCC
下游引物18:GATCATATCGACGCCAGCCTCATCAAAAATGCG
L59F
上游引物19:GCTGCCAACGTTGTGTTTGGTCGCGATACCACC
下游引物20:AAACACAACGTTGGCAGCGGAATCACCAACAAG
V89I
上游引物21:AAGCGTGCGCTTGTGATCGTTGATCTGCCGTTT
下游引物22:GATCACAAGCGCACGCTTCGTAGCGATCGTCAC
A185S
上游引物23:CAGGCGGGTGCGTTTTCCGTTGTGTTGGAGATG
下游引物24:GGAAAACGCACCCGCCTGCTCCAACGCGCGGGC
单位点突变菌株的琼脂糖凝胶电泳图参见图2和图3。
实施例4:构建双突变位点文库
通过Swiss-Model对谷氨酸棒杆菌来源的panB进行3D模型的构建,并使用AutoDock分子对接软件将KPHMT(panB)的模型和底物小分子α-酮异戊酸和5,10-亚甲基四氢叶酸进行分子对接,选取panB基因上的关键位点进行突变。设计引物构建双突变位点基因工程菌。
设计引物如下:
V21E/Q24A
上游引物25:GCTAAAGAGAACGGCGCTAAAGTTTCGGTTCTC
下游引物26:AGCGCCGTTCTCTTTAGCTTCGCGGAAATGACG
Q24A/K25A
上游引物27:AAAGTAAACGGCGCTGCTGTTTCGGTTCTCACC
下游引物28:AGCAGCGCCGTTTACTTTAGCTTCGCGGAAATG
Q24A/S27A
上游引物29:AACGGCGCTAAAGTTGCTGTTCTCACCAGCTAT
下游引物30:AGCAACTTTAGCGCCGTTTACTTTAGCTTCGCG
L59F/R61F
上游引物31:AACGTTGTGTTTGGTTTTGATACCACCTTGTCG
下游引物32:AAAACCAAACACAACGTTGGCAGCGGAATCACC
双位点突变菌株的琼脂糖凝胶电泳图参见图3。
实施例5:构建三突变位点
通过Swiss-Model对谷氨酸棒杆菌来源的panB进行3D模型的构建,并使用AutoDock分子对接软件将KPHMT(panB)的模型和底物小分子α-酮异戊酸和5,10-亚甲基四氢叶酸进行分子对接,选取panB基因上的关键位点进行突变。设计引物构建三突变位点基因工程菌。
设计引物如下:
V21E/V26N/S27A
上游引物33:GAGAACGGCCAGAAAACCGCTGTTCTCACCAGC
下游引物34:AGCGGTTTTCTGGCCGTTCTCTTTAGCTTCGCG
K25A/V26N/S27A
上游引物35:AACGGCCAGGCTACCGCTGTTCTCACCAGCTAT
下游引物36:AGCGGTAGCCTGGCCGTTTACTTTAGCTTCGCG
三位点突变菌株的琼脂糖凝胶电泳图参见图3。
实施例6:突变体发酵菌株构建
DPA-11-pTrc99A-panBpanC菌株构建:
首先消除pTrc99A质粒,再以空质粒pTrc99A为模板,通过PCR扩增获取线性化质粒片段,并分别获得panB及panC目的片段,将质粒框架和目的片段无缝连接,连接好的产物及时转化到E.coli DH5α感受态中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,获得pTrc99A-panBpanC菌株;
将构建好的pTrc99A-panBpanC菌株的质粒导入W3110Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk感受态细胞中,涂布于含Amp抗性的LB固体平板中,倒置于37℃恒温培养箱中过夜培养,即为所述大肠杆菌DPA-11-pTrc99A-panBpanC。
以E.coli DH5-α为克隆宿主,用于构建DPA-11-panBmuts突变质粒,对于实验室已构建好的含有panB(Corynebacterium glutamicum ATCC 13032)的产D-泛酸基因工程DPA-11-pTrc99A-panBpanC,设计相关突变位点引物,以DPA-11-pTrc99A-panBpanC质粒为模板,通过PCR技术得到一系列酮泛解酸羟甲基转移酶突变体分别获得突变体DPA-11-panB-E18A,DPA-11-panB-K20A,DPA-11-panB-V21E,DPA-11-panB-Q24A,DPA-11-panB-K25A,DPA-11-panB-V26N,DPA-11-panB-S27A,DPA-11-panB-M47I,DPA-11-panB-L48I,DPA-11-panB-L59F,DPA-11-panB-V89I,DPA-11-panB-A185S,DPA-11-panB-V21E/Q24A,DPA-11-panB-Q24A/K25A,DPA-11-panB-Q24A/S27A,DPA-11-panB-L59F/R61F,DPA-11-panB-V21E/V26N/S27A,DPA-11-panB-K25A/V26N/S27A。
以DPA-11-pTrc99A质粒为模板,通过PCR引入定点突变,PCR反应程序如下:95℃5min,95℃30s,58℃30s,72℃4min(2kbp/min),重复30个循环;72℃继续延伸10min。将PCR产物用DpnI于37℃处理15min,灭活后转化至E.coli DH5α感受态细胞中,涂布于含终浓度50mg/L氨苄抗性的LB固体平板上,37℃培养12h后,随机挑取单菌落进行测序,测序结果正确并得到重组质粒。将突变后的重组质粒DPA-11-panB-muts转化至DPA-11无质粒基因工程菌感受态细胞中,涂Amp+LB平板,37℃培养过夜,挑选单菌落,接种到含有LB液体培养基的试管中,37℃培养过夜,离心收集菌体,抽提质粒,基因测序确定突变正确,得到工程菌。
实施例7:突变菌株发酵
从突变后的基因工程菌DPA-11-panB-muts的LB平板上挑选单克隆,接种到5ml LB试管中,37℃培养过夜;按5%v/v比例接种到含有20ml M-1发酵培养基的250ml摇瓶中,30℃、180rpm培养48小时,离心获得菌体发酵液。培养基配方如下:
LB培养基(g/L):10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,pH7.2(LB固体培养基另加20g/L琼脂粉)。
M-1培养基(g/L):葡萄糖20g/L,硫酸铵16g/L,酵母粉2g/L,KH2PO4 0.8g/L,MgSO40.5g/L,CaCO3 15g/L,盐溶液1mL(其中包括CuCl2 10g/L,FeSO4·7H2O 10g/L,ZnSO4·H2O1g/L,CuSO4 0.2g/L,NiCl2·7H2O 0.02g/L),500mL摇瓶中装20mL培养基。接种时加入0.3gCaCO3,IPTG(终浓度为0.1mM)和Amp抗性(终浓度50mg/L),L-异亮氨酸(终浓度20mg/L),VB1(终浓度5mg/L),VB12(终浓度2mg/L)。(注:D-Pa-11为L-异亮氨酸缺陷型,则需在摇培养时添L-异亮氨酸(终浓度20mg/L)。
LB培养基使用前在121℃高温高压灭菌20分钟;M-1培养基使用前在115℃高温高压灭菌15分钟。
实施例8:D-泛酸标样标准曲线图
称取D-泛酸钙对照品0.05g(准确到0.001g),置于一10mL容量瓶中,加水溶解并稀释至不同浓度(0,0.5,1,2,2.5g/L),制成系列标准工作溶液。后使用0.22μm有机滤膜过膜,用于高效液相色谱(HPLC)检测。检测α-酮异戊酸的高效液相色谱操作条件如下所述。
A流动相成分:95%超纯水、4.9%乙腈、0.1%磷酸,使用0.20μm微孔水系滤膜过膜并超生除气泡;
B色谱柱型号:ACQUITY UPLC BEH C18 column(100mm×2.1mm,1.7μm,Waters,UK);
C参数设置:进样量:10μL,柱温:30℃,流速:0.9mL/min,检测波长:200nm。
D-泛酸标准曲线图参见图4。
实施例9:HPLC检测突变菌株D-泛酸产量
产物D-泛酸的HPLC测定条件:使用赛默飞液相Ultimate型号进行检测,ACQUITYUPLC BEH C18 column(100mm×2.1mm,1.7μm,waters,UK),流动相成分:95%超纯水、4.9%乙腈、0.1%磷酸,使用0.20μm微孔水系滤膜过膜并超声除气泡;流速0.9ml/min,柱温箱30℃,检测波长,200nm,进样量10ul,检测时长25min。
计算D-泛酸产率。结果见表1。
表1:各突变体D-泛酸产率
突变体DPA-11-panB-E18A,DPA-11-panB-K20A,DPA-11-panB-V21E,DPA-11-panB-Q24A,DPA-11-panB-K25A,DPA-11-panB-V26N,DPA-11-panB-S27A,DPA-11-panB-M47I,DPA-11-panB-L48I,DPA-11-panB-L59F,DPA-11-panB-V89I,DPA-11-panB-A185S,DPA-11-panB-V21E/Q24A,DPA-11-panB-Q24A/K25A,DPA-11-panB-Q24A/S27A,L59F/R61F,DPA-11-panB-V21E/V26N/S27A,DPA-11-panB-K25A/V26N/S27A通过生物发酵法提高了D-泛酸的产率,分别为3.1%,5.2%,10.1%,8.4%,6.2%,6.9%,11.6%,5.2%,6.4%,6.9%,3.0%,28.2%,12.3%,13.2%,5.2%,3.3%,4.9%,4.8%。具有工业化开发和应用前景。
D-泛酸HPLC检测图参见图5。
D-泛酸产量柱形图参见图6。
实施例10:突变菌株OD600检测
发酵结束后,取1mL菌液,在常温状态下12000rpm离心1min,转移上清至1.5mL Ep管冰箱并放入冰箱保存用于检测,菌体使用蒸馏水和20%乙酸(w/w)清洗后,稀释20~30倍测OD600,根据稀释倍数换算后得到发酵液实际OD600值(突变菌株OD600柱形图参见图6)。
突变菌株OD600。结果见表2。
表2:突变菌株OD600值
序列表
<110> 浙江工业大学
<120> 一种高产D-泛酸基因工程菌及其构建与应用
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 271
<212> PRT
<213> 未知(Unknown)
<400> 1
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Leu Ser Ile Thr Leu Asp Glu Met Ile Val Leu Ala Lys Ala Val Thr
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Ile Ala Thr Lys Arg Ala Leu Val Val Val Asp Leu Pro Phe Gly Thr
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Tyr Glu Val Ser Pro Asn Gln Ala Val Glu Ser Ala Ile Arg Val Met
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Arg Glu Thr Gly Ala Ala Ala Val Lys Ile Glu Gly Gly Val Glu Ile
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Ala Gln Thr Ile Arg Arg Ile Val Asp Ala Gly Ile Pro Val Val Gly
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His Ile Gly Tyr Thr Pro Gln Ser Glu His Ser Leu Gly Gly His Val
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Gly Ile Gly Ala Gly Asn Gly Thr Asp Gly Gln Val Leu Val Trp Gln
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Asp Ala Phe Gly Leu Asn Arg Gly Lys Lys Pro Arg Phe Val Arg Glu
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Tyr Ala Thr Leu Gly Asp Ser Leu His Asp Ala Ala Gln Ala Tyr Ile
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Ala Asp Ile His Ala Gly Thr Phe Pro Gly Glu Ala Glu Ser Phe
260 265 270
<210> 2
<211> 816
<212> DNA
<213> 未知(Unknown)
<400> 2
atgcccatgt caggcattga tgcaaagaaa atccgcaccc gtcatttccg cgaagctaaa 60
gtaaacggcc agaaagtttc ggttctcacc agctatgatg cgctttcggc gcgcattttt 120
gatgaggctg gcgtcgatat gctccttgtt ggtgattccg ctgccaacgt tgtgctgggt 180
cgcgatacca ccttgtcgat caccttggat gagatgattg tgctggccaa ggcggtgacg 240
atcgctacga agcgtgcgct tgtggtggtt gatctgccgt ttggtaccta tgaggtgagc 300
ccaaatcagg cggtggagtc cgcgatccgg gtcatgcgtg aaacgggtgc ggctgcggtg 360
aagatcgagg gtggcgtgga gatcgcgcag acgattcgac gcattgttga tgctggaatt 420
ccggttgtcg gccacatcgg gtacaccccg cagtccgagc attccttggg cggccacgtg 480
gttcagggtc gtggcgcgag ttctggaaag ctcatcgccg atgcccgcgc gttggagcag 540
gcgggtgcgt ttgcggttgt gttggagatg gttccagcag aggcagcgcg cgaggttacc 600
gaggatcttt ccatcaccac tatcggaatc ggtgccggca atggcacaga tgggcaggtt 660
ttggtgtggc aggatgcctt cggcctcaac cgcggcaaga agccacgctt cgtccgcgag 720
tacgccacct tgggcgattc cttgcacgac gccgcgcagg cctacatcgc cgatatccac 780
gcgggtacct tcccaggcga agcggagtcc ttttaa 816
<210> 3
<211> 271
<212> PRT
<213> 未知(Unknown)
<400> 3
Met Pro Met Ser Gly Ile Asp Ala Lys Lys Ile Arg Thr Arg His Phe
1 5 10 15
Arg Glu Ala Lys Val Asn Gly Gln Lys Val Ser Val Leu Thr Ser Tyr
20 25 30
Asp Ala Leu Ser Ala Arg Ile Phe Asp Glu Ala Gly Val Asp Met Leu
35 40 45
Leu Val Gly Asp Ser Ala Ala Asn Val Val Leu Gly Arg Asp Thr Thr
50 55 60
Leu Ser Ile Thr Leu Asp Glu Met Ile Val Leu Ala Lys Ala Val Thr
65 70 75 80
Ile Ala Thr Lys Arg Ala Leu Val Val Val Asp Leu Pro Phe Gly Thr
85 90 95
Tyr Glu Val Ser Pro Asn Gln Ala Val Glu Ser Ala Ile Arg Val Met
100 105 110
Arg Glu Thr Gly Ala Ala Ala Val Lys Ile Glu Gly Gly Val Glu Ile
115 120 125
Ala Gln Thr Ile Arg Arg Ile Val Asp Ala Gly Ile Pro Val Val Gly
130 135 140
His Ile Gly Tyr Thr Pro Gln Ser Glu His Ser Leu Gly Gly His Val
145 150 155 160
Val Gln Gly Arg Gly Ala Ser Ser Gly Lys Leu Ile Ala Asp Ala Arg
165 170 175
Ala Leu Glu Gln Ala Gly Ala Phe Ser Val Val Leu Glu Met Val Pro
180 185 190
Ala Glu Ala Ala Arg Glu Val Thr Glu Asp Leu Ser Ile Thr Thr Ile
195 200 205
Gly Ile Gly Ala Gly Asn Gly Thr Asp Gly Gln Val Leu Val Trp Gln
210 215 220
Asp Ala Phe Gly Leu Asn Arg Gly Lys Lys Pro Arg Phe Val Arg Glu
225 230 235 240
Tyr Ala Thr Leu Gly Asp Ser Leu His Asp Ala Ala Gln Ala Tyr Ile
245 250 255
Ala Asp Ile His Ala Gly Thr Phe Pro Gly Glu Ala Glu Ser Phe
260 265 270
<210> 4
<211> 816
<212> DNA
<213> 未知(Unknown)
<400> 4
atgcccatgt caggcattga tgcaaagaaa atccgcaccc gtcatttccg cgaagctaaa 60
gtaaacggcc agaaagtttc ggttctcacc agctatgatg cgctttcggc gcgcattttt 120
gatgaggctg gcgtcgatat gctccttgtt ggtgattccg ctgccaacgt tgtgctgggt 180
cgcgatacca ccttgtcgat caccttggat gagatgattg tgctggccaa ggcggtgacg 240
atcgctacga agcgtgcgct tgtggtggtt gatctgccgt ttggtaccta tgaggtgagc 300
ccaaatcagg cggtggagtc cgcgatccgg gtcatgcgtg aaacgggtgc ggctgcggtg 360
aagatcgagg gtggcgtgga gatcgcgcag acgattcgac gcattgttga tgctggaatt 420
ccggttgtcg gccacatcgg gtacaccccg cagtccgagc attccttggg cggccacgtg 480
gttcagggtc gtggcgcgag ttctggaaag ctcatcgccg atgcccgcgc gttggagcag 540
gcgggtgcgt tttccgttgt gttggagatg gttccagcag aggcagcgcg cgaggttacc 600
gaggatcttt ccatcaccac tatcggaatc ggtgccggca atggcacaga tgggcaggtt 660
ttggtgtggc aggatgcctt cggcctcaac cgcggcaaga agccacgctt cgtccgcgag 720
tacgccacct tgggcgattc cttgcacgac gccgcgcagg cctacatcgc cgatatccac 780
gcgggtacct tcccaggcga agcggagtcc ttttaa 816
<210> 5
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 5
acccgtcatt tccgcgctgc taaagtaaac ggc 33
<210> 6
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 6
agcgcggaaa tgacgggtgc ggattttctt tgc 33
<210> 7
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 7
catttccgcg aagctgctgt aaacggccag aaa 33
<210> 8
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 8
agcagcttcg cggaaatgac gggtgcggat ttt 33
<210> 9
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 9
ttccgcgaag ctaaagagaa cggccagaaa gtt 33
<210> 10
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 10
ctctttagct tcgcggaaat gacgggtgcg gat 33
<210> 11
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 11
gctaaagtaa acggcgctaa agtttcggtt ctc 33
<210> 12
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 12
agcgccgttt actttagctt cgcggaaatg acg 33
<210> 13
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 13
aaagtaaacg gccaggctgt ttcggttctc acc 33
<210> 14
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 14
agcctggccg tttactttag cttcgcggaa atg 33
<210> 15
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 15
gtaaacggcc agaaaacctc ggttctcacc agc 33
<210> 16
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 16
ggttttctgg ccgtttactt tagcttcgcg gaa 33
<210> 17
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 17
aacggccaga aagttgctgt tctcaccagc tat 33
<210> 18
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 18
agcaactttc tggccgttta ctttagcttc gcg 33
<210> 19
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 19
gaggctggcg tcgatatcct ccttgttggt gat 33
<210> 20
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 20
gatatcgacg ccagcctcat caaaaatgcg cgc 33
<210> 21
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 21
gctggcgtcg atatgatcct tgttggtgat tcc 33
<210> 22
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 22
gatcatatcg acgccagcct catcaaaaat gcg 33
<210> 23
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 23
gctgccaacg ttgtgtttgg tcgcgatacc acc 33
<210> 24
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 24
aaacacaacg ttggcagcgg aatcaccaac aag 33
<210> 25
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 25
aagcgtgcgc ttgtgatcgt tgatctgccg ttt 33
<210> 26
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 26
gatcacaagc gcacgcttcg tagcgatcgt cac 33
<210> 27
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 27
caggcgggtg cgttttccgt tgtgttggag atg 33
<210> 28
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 28
ggaaaacgca cccgcctgct ccaacgcgcg ggc 33
<210> 29
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 29
gctaaagaga acggcgctaa agtttcggtt ctc 33
<210> 30
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 30
agcgccgttc tctttagctt cgcggaaatg acg 33
<210> 31
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 31
aaagtaaacg gcgctgctgt ttcggttctc acc 33
<210> 32
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 32
agcagcgccg tttactttag cttcgcggaa atg 33
<210> 33
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 33
aacggcgcta aagttgctgt tctcaccagc tat 33
<210> 34
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 34
agcaacttta gcgccgttta ctttagcttc gcg 33
<210> 35
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 35
aacgttgtgt ttggttttga taccaccttg tcg 33
<210> 36
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 36
aaaaccaaac acaacgttgg cagcggaatc acc 33
<210> 37
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 37
gagaacggcc agaaaaccgc tgttctcacc agc 33
<210> 38
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 38
agcggttttc tggccgttct ctttagcttc gcg 33
<210> 39
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 39
aacggccagg ctaccgctgt tctcaccagc tat 33
<210> 40
<211> 33
<212> DNA
<213> 未知(Unknown)
<400> 40
agcggtagcc tggccgttta ctttagcttc gcg 33
Claims (5)
1.一种高产D-泛酸基因工程菌,以大肠杆菌DPA-11-pTrc99A-panBpanC为底盘菌,将所述底盘菌中来源于谷氨酸棒杆菌的panB基因进行突变,使得由该panB基因编码的如SEQ IDNO:1所示氨基酸序列的第185位丙氨酸突变为丝氨酸;
所述大肠杆菌DPA-11-pTrc99A-panBpanC按如下方法构建获得:
(1)应用CRISPR-Cas9基因编辑技术将底盘菌E .coli W3110基因组中的panC、panB、panE和ilvC基因的启动子替换为trc启动子,得到菌株E .coli W3110 Trc-panCpanEpanBilvC ;
(2)应用CRISPR-Cas9基因编辑技术在菌株E .coli W3110 Trc-panCpanEpanBilvC 基因组中修复ilvG基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG* ;
(3)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*中avtA基因敲除,得到菌株E .coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA;
(4)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA上ilvE基因的起始密码子ATG替换为GTG,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*;
(5)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*中coaA编码的酶第106位氨基酸由丙氨酸突变为精氨酸,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*;
(6)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA* 中ilvA基因敲除,得到菌株E .coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA;
(7)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA 中lpd基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd;
(8)应用CRISPR-Cas9基因编辑技术敲除菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd中的glk基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk;
(9)以质粒pTrc99A为模板,通过PCR扩增获取线性化质粒片段,并分别获得panB及panC目的片段,将质粒框架和目的片段无缝连接,连接好的产物及时转化到E. coli DH5α感受态中,涂布于含Amp抗性的LB固体平板中,倒置于37 ℃恒温培养箱中过夜培养,获得pTrc99A-panBpanC菌株;所述panB来源于谷氨酸棒杆菌,其核苷酸序列如SEQ ID NO:2所示;
(10)将构建好的pTrc99A-panBpanC菌株的质粒导入E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk感受态细胞中,涂布于含Amp抗性的 LB 固体平板中,倒置于37 ℃恒温培养箱中过夜培养,即为所述大肠杆菌DPA-11-pTrc99A-panBpanC。
2.构建权利要求1所述高产D-泛酸基因工程菌的方法,所述方法包括:以大肠杆菌DPA-11-pTrc99A-panBpanC为底盘菌,将所述底盘菌中来源于谷氨酸棒杆菌的panB基因进行突变,使得由该panB基因编码的如SEQ ID NO:1所示氨基酸序列的第185位丙氨酸突变为丝氨酸,获得所述高产D-泛酸基因工程菌。
3.如权利要求2所述的方法,其特征在于所述大肠杆菌DPA-11-pTrc99A-panBpanC按如下方法构建获得:
(1)应用CRISPR-Cas9基因编辑技术将底盘菌E .coli W3110基因组中的panC、panB、panE和ilvC基因的启动子替换为trc启动子,得到菌株E .coli W3110 Trc-panCpanEpanBilvC ;
(2)应用CRISPR-Cas9基因编辑技术在菌株E .coli W3110 Trc-panCpanEpanBilvC 基因组中修复ilvG基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG* ;
(3)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*中avtA基因敲除,得到菌株E .coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA;
(4)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA上ilvE基因的起始密码子ATG替换为GTG,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*;
(5)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*中coaA编码的酶第106位氨基酸由丙氨酸突变为精氨酸,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*;
(6)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA* 中ilvA基因敲除,得到菌株E .coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA;
(7)应用CRISPR-Cas9基因编辑技术将菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA 中lpd基因的启动子替换为trc启动子,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd;
(8)应用CRISPR-Cas9基因编辑技术敲除菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd中的glk基因,得到菌株E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk;
(9)以质粒pTrc99A为模板,通过PCR扩增获取线性化质粒片段,并分别获得panB及panC目的片段,将质粒框架和目的片段无缝连接,连接好的产物及时转化到E. coli DH5α感受态中,涂布于含Amp抗性的LB固体平板中,倒置于37 ℃恒温培养箱中过夜培养,获得pTrc99A-panBpanC菌株;所述panB来源于谷氨酸棒杆菌,其核苷酸序列如SEQ ID NO:2所示;
(10)将构建好的pTrc99A-panBpanC菌株的质粒导入E.coli W3110 Trc-panCpanEpanBilvC/ilvG*/ΔavtA/ilvE*/coaA*/ΔilvA/Trc-lpd/Δglk感受态细胞中,涂布于含Amp抗性的 LB 固体平板中,倒置于37 ℃恒温培养箱中过夜培养,即为所述大肠杆菌DPA-11-pTrc99A-panBpanC。
4.权利要求1所述高产D-泛酸基因工程菌在微生物发酵制备D-泛酸中的应用。
5.如权利要求4所述的应用,其特征在于所述应用为:将所述高产D-泛酸基因工程菌接种至发酵培养基,进行摇瓶发酵制备D-泛酸。
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