CN113388564B - 一种o-乙酰-l-高丝氨酸生产菌、构建方法及应用 - Google Patents
一种o-乙酰-l-高丝氨酸生产菌、构建方法及应用 Download PDFInfo
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Abstract
本发明涉及一种O‑乙酰‑L‑高丝氨酸(OAH)生产菌、构建方法及应用。所述O‑乙酰‑L‑高丝氨酸(OAH)生产菌是以E.coli W3110为底盘菌,将其基因组中的metA、metB、thrB和metJ基因敲除,并增强外源metA基因的表达所得。本发明构建所得OAH生产菌在发酵过程中,能实现发酵液中OAH的积累,OAH摇瓶产量3.58g/L,补料发酵产量16.35g/L,为后续高产OAH工程菌的构建奠定了基础。
Description
(一)技术领域
本发明属于代谢工程领域,具体涉及一种O-乙酰-L-高丝氨酸生产菌、构建方法及应用。
(二)背景技术
蛋氨酸是人类和其他动物生活的一种必需氨基酸,是生物体内蛋白质的组成部分。目前,L-蛋氨酸在食品,药品和饲料添加剂中起着重要的作用,因此具有广阔的市场应用前景。现阶段,蛋氨酸的合成方法主要有化学法、酶法和微生物发酵法。其中,化学法和酶法的合成前体昂贵且污染严重,限制了蛋氨酸的工业生产和应用;由于微生物体内蛋氨酸的合成存在多分支途径及多水平的调控体系,使微生物合成蛋氨酸陷入产量较低的瓶颈。因此,人们致力于寻找更为理想的蛋氨酸生产方法。
目前,通过微生物发酵生产蛋氨酸前体结合酶催化法逐渐引起人们的关注。O-乙酰-L-高丝氨酸(OAH)是生产高丝氨酸内酯,蛋氨酸和其他有价值化合物的重要平台代谢中间体。工业上可以以OAH为前体,通过酶法工艺与甲硫醇结合合成蛋氨酸。目前,以O-琥珀酰-L-高丝氨酸(OSH)(O-乙酰-L-高丝氨酸的结构类似物)与甲硫醇直接反应生产L-蛋氨酸和琥珀酸的路线,已在马来西亚成功运行了数年。因此,构建基因工程菌株生产OAH,再通过酶法合成蛋氨酸具有一定的应用前景。
(三)发明内容
本发明的目的是通过代谢工程技术,提供一种O-乙酰-L-高丝氨酸(OAH)生产菌、构建方法及应用。
为实现本发明的上述目的,本发明采用的技术方案:
一种O-乙酰-L-高丝氨酸生产菌,由如下方法构建获得:
(1)以E.coli W3110为底盘菌,将其基因组中的metA、metB、thrB和metJ基因敲除,得到重组菌株W3110△metA△metB△thrB△metJ,记为OAHL4;
(2)将源自Bacillus cereus的基因metA(具有metX的催化特性)整合入质粒pACYCDuet-1中,得到质粒pACYCDuet-metA;将质粒pACYCDuet-metA导入步骤(1)所述基因工程菌OAHL4中,得到所述O-乙酰-L-高丝氨酸生产菌(记为OAHL5)。
优选的,所述源自Bacillus cereus的基因metA核苷酸序列如SEQ ID No.1所示。
本发明还涉及构建所述的O-乙酰-L-高丝氨酸生产菌的方法,其特征在于所述方法如下:
(1)运用CRISPR-Cas9基因编辑技术,将底盘菌E.coli W3110基因组中的metA、metB、thrB和metJ基因敲除,得到重组菌株W3110△metA△metB△thrB△metJ,记为OAHL4;
(2)将核苷酸序列如SEQ ID No.1所示的外源基因metA整合入质粒pACYCDuet-1中,得到质粒pACYCDuet-metA;将质粒pACYCDuet-metA导入步骤(1)所述重组菌OAHL4中,得到所述O-乙酰-L-高丝氨酸生产菌(记为OAHL5)。
本发明还涉及所述O-乙酰-L-高丝氨酸生产菌在微生物发酵制备O-乙酰-L-高丝氨酸中的应用。
具体的,所述应用为:将所述O-乙酰-L-高丝氨酸生产菌接种至发酵培养基中,于28~32℃、100~500rpm条件下进行发酵培养36~48h,发酵结束后取发酵液上清分离纯化得到O-乙酰-L-高丝氨酸;所述发酵培养基组成如下:葡萄糖10~30g/L、硫酸铵10~20g/L、酵母浸粉1~5g/L、KH2PO4 1~5g/L、MgSO4 0.1~2.0g/L、CaCO3 10~20g/L、微量金属盐溶液0.5~5mL/L,pH 6.5~7.0,溶剂为去离子水;微量金属盐溶液组成为:10g/L CuCl2、10g/L FeSO4·7H2O、1g/L ZnSO4·7H2O、0.20g/L CuSO4、0.02g/L NiCl2·7H2O,溶剂为去离子水。
优选的,所述发酵培养基组成如下:葡萄糖20g/L,硫酸铵16g/L,酵母粉2g/L,KH2PO4 1g/L,MgSO4 0.2g/L,CaCO3 15g/L,微量金属盐溶液1mL/L,溶剂为去离子水。
大肠杆菌中OAH生物合成途径及本发明涉及的基因工程改造示意图参见图1。首先,以E.coli W3110为底盘细胞,敲除基因metA,阻断OAH合成前体高丝氨酸的降解;其次,敲除基因metB,阻断竞争性蛋氨酸合成支路;然后再敲除基因thrB,阻断竞争性苏氨酸合成支路;敲除metJ,消除转录调节抑制;同时,由于大肠杆菌缺少基因metX,导致本身不能合成OAH,研究发现来自Bacillus cereus的metA基因具有metX活性,所以将外源基因metA(源自Bacillus cereus)整合入质粒pACYCDuet-1中,得到质粒pACYCDuet-metA,并将其导入改造菌株中,最终得到OAH生产菌株。
通常,所述基因工程菌株发酵前,先接种至LB培养基中,于温度37℃、转速200rpm的摇床上过夜培养,然后以体积浓度5~15%接种量接种到发酵培养基中培养。
本发明有益效果主要体现在:本发明通过敲除基因metA、metB、thrB和metJ阻断竞争性途径和消除转录调节,再通过增强外源metA基因的表达,得到了OAH生产菌株,且该菌株在发酵过程中,能实现OAH的积累,OAH摇瓶产量达3.58g/L,5L发酵罐补料发酵产量打16.35g/L(,为后续高产OAH工程菌的构建奠定了基础。
(四)附图说明
图1:大肠杆菌中OAH生物合成途径及本发明涉及的基因工程改造示意图;
图2:质粒pACYCDuet-metA图谱。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
以下实施例中,所述卡那霉素(kana)的使用终浓度为50ng/μL。所述盐酸壮观霉素(SD)的使用终浓度为50ng/μL,所述氯霉素的使用终浓度为25ng/μL。实施例中所用大肠杆菌感受态细胞为购买的商用大肠杆菌DH5α,重组反应所用的一步法定向克隆无缝克隆试剂盒购于诺唯赞公司,但不局限于此公司。
实施例1:敲除基因metA,构建菌株OAHL1
构建OAH生产菌株采用的基因编辑方法参照文献(Zhang,B.,et al.,Metabolicengineering of Escherichia coli for D-pantothenic acid production.Food Chem,2019.294:p.267-275.),具体方法如下:
(1)pTarget-gRNA质粒突变
设计定点突变引物,将pTarget上20bp同源序列突变成靶基因metA的PAM位点前的20bp序列。以pTarget为模板,ΔmetA-pT F和ΔmetA-pT R为引物,PCR扩增pTarget突变质粒,扩增体系如下:
PCR扩增结束后,利用琼脂糖凝胶电泳检测PCR产物,然后向PCR产物中加入0.5μL的DpnI,37℃消化1h,取消化产物10μL加入到100μL的DH5α感受态细胞中进行转化实验,然后将培养液涂布于LB固体培养基(SD抗性),37℃培养过夜。挑取单菌落接种于10mL的LB液体培养基(SD抗性)中,培养18h以上,取菌液送测序并提取突变质粒pTarget-metA-g备用。
(2)PCR扩增donor DNA和线性化pTarget-metA-g质粒片段
以大肠杆菌基因组为模板,分别以ΔmetA-P1、ΔmetA-P2和ΔmetA-P3、ΔmetA-P4为引物PCR扩增出靶基因上下游各约500bp序列作为同源臂(donor DNA)。以p T+D CF com、p T+D CR com为引物,Target-metA-g为模板,PCR扩增Target-metA-g线性化质粒片段。扩增体系如下:
PCR扩增结束后,利用琼脂糖凝胶电泳检测PCR产物,然后向线性化Target-metA-g质粒片段中加入1μL的DpnI,37℃消化1h。然后使用DNA清洁试剂盒对消化产物和PCR扩增的donor DNA进行clean up,并检测清洁后产物的DNA浓度,-20℃保存备用。
(3)一步克隆连接线性化Target-metA-g质粒片段和donor DNA
取(2)中清洁后的产物,根据线性化Target-metA-g质粒片段和donor DNA的浓度,利用一步法定向克隆无缝克隆试剂盒进行重组反应。取一步克隆产物10μL加入到100μL的DH5α感受态细胞中进行转化实验,然后将培养液涂布于LB固体培养基(SD抗性),37℃培养过夜。待长出单菌落,挑取单菌落接种10mL的LB液体培养基(SD抗性),送测序并提取质粒pTarget-metA-pdg备用。
(4)pTarget-metA-pdg的电击转化
制备含pCas质粒的E.coli W3110菌株(实验室保藏)的电转感受态。取一只电转感受态,冰浴放置,在超净台中加入2μL pTarget-metA-pdg,用移液器轻柔混匀,冰浴1min;用移液器将混匀液转移至预冷的2mm电击杯中(电击杯需提前在超净台风干),冰浴45s。用纸巾擦干电击杯外侧水雾,置于电转仪中,使用Eco 2档电击。在超净台中向电击杯凹槽加入1mL预冷LB培养基,倾斜电击杯,从电击杯口吸取全部菌液,转移至2mL无菌EP管中。30℃,180rpm复苏2.5h以上;取200μL涂布于LB固体培养基(SD+kana抗性),30℃培养过夜。
(5)阳性克隆筛选和验证
在靶标基因上游同源臂外侧100bp处设计正向验证引物ΔmetA-VF,在靶标基因下游同源臂外侧100bp处设计反向验证引物ΔmetA-VR,并以此引物为正反引物,挑取克隆子作为模板,进行菌落PCR,同时以原基因组为模板作阴性对照。菌落PCR有条带,大小正确视为阳性。
(6)pTarget-pdg与pCas消除与测序验证
将阳性克隆接种于10mL LB试管(kana抗性),并加入10μL IPTG母液,30℃,180rpm培养12h。取过夜培养菌液划线于LB固体培养基(kana抗性),30℃培养过夜。取过夜划线平板,将单菌落编号,并挑取编号的部分单菌落,划线于LB固体培养基(SD抗性)上所对应的区域,37℃培养过夜。在LB固体培养基(SD抗性)所对应的区域不能生长的单菌落为pTarget-metA-pdg成功消除的克隆。
将pTarget-metA-pdg成功消除的克隆接种于10mL LB试管(无抗性),37℃,180rpm培养12h。取过夜培养菌液,划线于LB固体培养基(无抗性),37℃培养过夜。取过夜培养菌液,将单菌落编号,并挑取编号的部分单菌落,划线于LB固体培养基(kana抗性)上所对应的区域,30℃培养过夜。在LB固体培养基(kana抗性)所对应的区域不能生长的单菌落为pCas成功消除的克隆。
(7)阳性克隆测序验证
挑取pTarget-pdg与pCas均成功消除的克隆作为菌落PCR模板,以验证引物进行菌落PCR,菌落PCR产物送测序,验证阳性克隆,得到菌株OAHL1。
实施例2:敲除基因metB,构建菌株OAHL2
(1)pTarget-gRNA质粒突变
以pTarget为模板,ΔmetB-pT F和ΔmetB-pT R为引物,PCR扩增pTarget突变质粒。PCR产物经消化处理后,转化进入DH5α感受态细胞,经测序验证,得到突变质粒pTarget-metB-g。
(2)构建质粒pTarget-metB-pdg
以ΔmetB-P1、ΔmetB-P2和ΔmetB-P3、ΔmetB-P4为引物PCR扩增靶基因上下游donor DNA。同时以p T+D CF com、p T+D CR com为引物,pTarget-metB-g为模板,PCR扩增线性化pTarget-metB-g质粒片段。DpnI消化处理PCR扩增的pTarget-metB-g线性化质粒片段后,使用DNA清洁试剂盒对消化后的线性化pTarget-metB-g质粒片段和donor DNA进行clean up,再利用一步法定向克隆无缝克隆试剂盒进行重组反应,反应产物转化大肠杆菌感受态细胞后,经测序验证后得到质粒pTarget-metB-pdg。
(3)pTarget-metB-pdg的电击转化
制备含pCas质粒的OAHL1菌株的电转感受态。取一只电转感受态,在超净台中加入2μL pTarget-maeB-pdg,冰浴1min后转移至预冷的2mm电击杯中,冰浴45s。置于电转仪中进行电击。电击后立即向杯凹槽中加入1mL预冷LB培养基,倾斜电击杯,从电击杯口吸取全部菌液,转移至2mL无菌EP管中。30℃,180rpm复苏2.5h以上;取200μL涂布于LB固体培养基(SD+kana抗性),30℃培养过夜。
后续基因编辑、验证及质粒消除同实施例1(4-7),筛选阳性克隆得到菌株OAHL2。
实施例3:敲除基因thrB,构建菌株OAHL3
(1)pTarget-gRNA质粒突变
以pTarget为模板,ΔthrB-pT F和ΔthrB-pT R为引物,PCR扩增pTarget突变质粒。PCR产物经消化处理后,转化进入DH5α感受态细胞,经测序验证,得到突变质粒pTarget-thrB-g。
(2)构建质粒pTarget-thrB-pdg
以ΔthrB-P1、ΔthrB-P2和ΔthrB-P3、ΔthrB-P4为引物PCR扩增靶基因上下游donor DNA。同时以p T+D CF com、p T+D CR com为引物,pTarget-thrB-g为模板,PCR扩增线性化pTarget-thrB-g质粒片段。DpnI消化处理PCR扩增的线性化pTarget-thrB-g质粒片段后,使用DNA清洁试剂盒对消化后的线性化pTarget-thrB-g质粒片段和donor DNA进行clean up,再采用一步克隆法连接线性化pTarget-thrB-g质粒片段和donor DNA,经转化和测序验证后得到质粒pTarget-thrB-pdg。
(3)pTarget-thrB-pdg的电击转化
制备含pCas质粒的OAHL2菌株的电转感受态。取一只电转感受态,在超净台中加入2μL pTarget-thrB-pdg,冰浴1min后转移至预冷的2mm电击杯中,冰浴45s。置于电转仪中进行电击。电击后立即向杯凹槽中加入1mL预冷LB培养基,倾斜电击杯,从电击杯口吸取全部菌液,转移至2mL无菌EP管中。30℃,180rpm复苏2.5h以上;取200μL涂布于LB固体培养基(SD+kana抗性),30℃培养过夜。
后续基因编辑、验证及质粒消除同实施例1(4-7),筛选阳性克隆得到菌株OAHL3。
实施例4:敲除基因metJ,构建菌株OAHL4
(1)pTarget-gRNA质粒突变
以pTarget为模板,ΔmetJ-pT F和ΔmetJ-pT R为引物,PCR扩增pTarget突变质粒。PCR产物经消化处理后,转化进入DH5α感受态细胞,经测序验证,得到突变质粒pTarget-metJ-g。
(2)构建质粒pTarget-metJ-pdg
以ΔmetJ-P1、ΔmetJ-P2和ΔmetJ-P3、ΔmetJ-P4为引物PCR扩增靶基因上下游donor DNA。同时以p T+D CF com、p T+D CR com为引物,pTarget-metJ-g为模板,PCR扩增线性化pTarget-metJ-g质粒片段。DpnI消化处理PCR扩增的线性化pTarget-metJ-g质粒片段后,使用DNA清洁试剂盒对消化后的线性化pTarget-metJ-g质粒片段和donor DNA进行clean up,再采用一步克隆法连接线性化pTarget-metJ-g质粒片段和donor DNA,经转化和测序验证后得到质粒pTarget-metJ-pdg。
(3)pTarget-metJ-pdg的电击转化
制备含pCas质粒的OAHL3菌株的电转感受态。取一只电转感受态,在超净台中加入2μL pTarget-metJ-pdg,冰浴1min后转移至预冷的2mm电击杯中,冰浴45s。置于电转仪中进行电击。电击后立即向杯凹槽中加入1mL预冷LB培养基,倾斜电击杯,从电击杯口吸取全部菌液,转移至2mL无菌EP管中。30℃,180rpm复苏2.5h以上;取200μL涂布于LB固体培养基(SD+kana抗性),30℃培养过夜。
后续基因编辑、验证及质粒消除同实施例1(4-7),筛选阳性克隆得到菌株OAHL4。
实施例5:pACYCDuet-metA(Bc)质粒的构建
以pACYCDuet-1质粒(实验室保藏)为模板,pACYC-F、pACYC-R为引物,PCR扩增线性化质粒片段,同时以metA-F、metA-R为引物,蜡样枯草芽孢杆菌基因组为模板,扩增metA片段(序列如SEQ ID NO.1所示)。利用DpnI消化扩增的线性化质粒片段,然后使用clean up试剂盒对消化后的线性化pACYCDuet-1载体片段和metA片段进行清洁,再利用一步法定向克隆无缝克隆试剂盒进行重组反应,反应产物转化大肠杆菌感受态细胞后,涂布在含有氯霉素的LB平板上。以metA-VF、metA-VR为引物,菌落PCR验证单克隆,阳性克隆送测序公司测序,确定连接成功的质粒pACYCDuet-metA(Bc)。将构建得质粒pACYCDuet-metA(Bc)转化OAHL4,筛选阳性克隆得到菌株OAHL5。
表1:菌株基因型
表2:引物序列表
实施例6:工程菌株的发酵
菌种活化:取-80℃保藏菌种(OAHL5)划线接种于活化培养基(固体LB培养基)中,37℃培养过夜;
种子培养:用接种环挑取活化种子接种于装有10mL种子培养基(LB培养基)的试管中,37℃,200rpm培养过夜;
摇瓶发酵:按5%接种量接种种子液到装有20mL发酵培养基的500mL锥形瓶中,37℃,200rpm/min振荡培养,发酵周期48h;
发酵罐发酵:取种子培养基接种于3瓶含100mL LB培养基的500mL锥形瓶中,37℃,200rpm/min振荡培养12h。按15%的接种量将摇瓶种子液接入含2L发酵培养基的5L发酵罐中,发酵温度控制在30℃,转速500rpm/min,通过40%氨水控制发酵pH为6.8。发酵过程采用pH-star模式控制补料,每4h取样检测生物量、残糖和D-泛解酸的量。
摇瓶发酵培养基组成为:葡萄糖20g/L,硫酸铵16g/L,酵母粉2g/L,KH2PO41g/L,MgSO4 0.2g/L,CaCO3 15g/L,盐溶液1mL/L,其中CaCO3独立分装灭菌(每份0.3g)。接种时加入CaCO3和IPTG(终浓度0.025mM)。
补料培养基组成如下:葡萄糖500g/L、硫酸铵10g/L、酵母浸粉5g/L、KH2PO414g/L、MgSO4 8g/L、盐溶液1mL/L。
发酵结束后,OAH的检测使用日立L8080氨基酸分析仪,具体方法见L8080操作手册。
表3:基因工程菌发酵产OAH
通过基因工程操作后,基因工程菌OAHL5通过摇瓶发酵OAH产量达3.58g/L,补料发酵OAH产量达16.35g/L。因此,本发明所构建的OAH基因工程菌在发酵过程中能够实现发酵液中OAH的有效积累,为构建高产OAH的基因工程菌株的构建打下基础。
序列表
<110> 浙江工业大学
<120> 一种O-乙酰-L-高丝氨酸生产菌、构建方法及应用
<160> 35
<170> SIPOSequenceListing 1.0
<210> 1
<211> 918
<212> DNA
<213> 蜡状芽孢杆菌(Bacillus cereus)
<400> 1
atggaggcga agcacatgcc gatcattatc gacaaagatc tgccggcgcg taaggttctg 60
cagaaagaaa acattttcgt gatgaccaag gagcgtgcgg aaacccaaga catccgtgcg 120
ctgaagattg cgatcctgaa cctgatgccg accaaacagg ataccgaggc gcaactgctg 180
cgtctgattg gtaacacccc gctgcagctg gacgttcacc tgctgcacat ggagagccac 240
ctgagccgta acgtgaccca agaacacctg accagcttct acaagacctt tcgtgacatc 300
gagaacgaaa aattcgatgg tctgattatt accggtgcgc cggttgagac cctggcgttt 360
gaggaagtgg attactggga ggaactgaag cacattatgg aatatagcaa aaccaacgtt 420
accagcaccc tgcacatctg ctggggtgcg caggcgggcc tgtactatca ctacggtgtg 480
ccgaagtatc cgctgaagga gaaaatgttc ggcgtgtttg agcacgaagt ttgcgagcag 540
cacgtgaaac tgctgcaagg cttcgatgaa ctgttctttg cgccgcacag ccgtcacacc 600
gaggttcgtg aaaacgacat tcgtgaggtg aaggaactga ccctgctggc gaacagcgag 660
gaagcgggtg tgcacctggt tatcggtccg gaaggccgtc aggtttttgc gctgggccac 720
agcgagtaca gctgcgaaac cctgaagcaa gagtatgaac gtgaccgtga taaaggtctg 780
aacattgacg ttccgaagaa ctatttcaaa cacaacaacc cggatgagaa accgctggtg 840
cgttggcgta gccacggcaa cctgctgttt agcaactggc tgaactacta tgtgtaccaa 900
gaaaccccgt atatcctg 918
<210> 2
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 2
taatactagt ctttaccggg caacctttgc gttttagagc tagaaatagc 50
<210> 3
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 3
gctctaaaac gcaaaggttg cccggtaaag actagtatta tacctaggac 50
<210> 4
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 4
cggtgctttt tttgaattct ctagagtcgt taccaggtga atc 43
<210> 5
<211> 32
<212> DNA
<213> 未知(Unknown)
<400> 5
cttttttgct gagatactta atcctcttcg tc 32
<210> 6
<211> 27
<212> DNA
<213> 未知(Unknown)
<400> 6
taagtatctc agcaaaaaag agcggcg 27
<210> 7
<211> 47
<212> DNA
<213> 未知(Unknown)
<400> 7
gggtaataga tctaagcttc tgcaggttct ggtaaacctt ccacacc 47
<210> 8
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 8
gactgtcgaa caggcgatag 20
<210> 9
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 9
taatactagt ttcgcgctat gctgactttc gttttagagc tagaaatagc 50
<210> 10
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 10
gctctaaaac gaaagtcagc atagcgcgaa actagtatta tacctaggac 50
<210> 11
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 11
cggtgctttt tttgaattct ctagacacat acgccagatt cag 43
<210> 12
<211> 34
<212> DNA
<213> 未知(Unknown)
<400> 12
tcacagaaga aacctgatta cctcactaca tacg 34
<210> 13
<211> 30
<212> DNA
<213> 未知(Unknown)
<400> 13
taatcaggtt tcttctgtga tagtcgatcg 30
<210> 14
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 14
gggtaataga tctaagcttc tgcagtctaa gtccgcagga atc 43
<210> 15
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 15
gtgatagtgc ggtcatgg 18
<210> 16
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 16
taatactagt gcgccgcacg actgctacgg gttttagagc tagaaatagc 50
<210> 17
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 17
gctctaaaac ccgtagcagt cgtgcggcgc actagtatta tacctaggac 50
<210> 18
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 18
cggtgctttt tttgaattct ctagatgctt tagtattccc acg 43
<210> 19
<211> 28
<212> DNA
<213> 未知(Unknown)
<400> 19
acactcattt gtgatgaagt tccctggg 28
<210> 20
<211> 30
<212> DNA
<213> 未知(Unknown)
<400> 20
acttcatcac aaatgagtgt gattgcgcag 30
<210> 21
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 21
gggtaataga tctaagcttc tgcaggcaac gggtaagaaa gcc 43
<210> 22
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 22
cttatccggc ctacaagttc 20
<210> 23
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 23
taatactagt atggcgatga atgaacactg gttttagagc tagaaatagc 50
<210> 24
<211> 50
<212> DNA
<213> 未知(Unknown)
<400> 24
gctctaaaac cagtgttcat tcatcgccat actagtatta tacctaggac 50
<210> 25
<211> 44
<212> DNA
<213> 未知(Unknown)
<400> 25
cggtgctttt tttgaattct ctagactttc tggtatggat gtgg 44
<210> 26
<211> 31
<212> DNA
<213> 未知(Unknown)
<400> 26
agagtttcat gtcagactcc taacttccat g 31
<210> 27
<211> 32
<212> DNA
<213> 未知(Unknown)
<400> 27
ggagtctgac atgaaactct acaatctgaa ag 32
<210> 28
<211> 43
<212> DNA
<213> 未知(Unknown)
<400> 28
gggtaataga tctaagcttc tgcaggccca atgtacagaa cag 43
<210> 29
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 29
gaattgatga agttctccg 19
<210> 30
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 30
aggggttttt tgctgaaac 19
<210> 31
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 31
atttcctaat gcaggagtcg 20
<210> 32
<211> 41
<212> DNA
<213> 未知(Unknown)
<400> 32
cgactcctgc attaggaaat taatacgact cactataggg g 41
<210> 33
<211> 38
<212> DNA
<213> 未知(Unknown)
<400> 33
ggtttcagca aaaaacccct caaaaaaccc ctcaagac 38
<210> 34
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 34
ggtgtggatc tgcacttcta c 21
<210> 35
<211> 22
<212> DNA
<213> 未知(Unknown)
<400> 35
cggatatagt tcctcctttc ag 22
Claims (6)
1.一种O-乙酰-L-高丝氨酸生产菌,由如下方法构建获得:
(1)以E. coli W3110为底盘菌,将其基因组中的metA、metB、thrB和metJ基因敲除,得到重组菌株W3110 △metA△metB△thrB△metJ,记为OAHL4;
(2)将源自Bacillus cereus的基因metA整合入质粒pACYCDuet-1中,得到质粒pACYCDuet-metA;将质粒pACYCDuet-metA导入步骤(1)所述重组菌OAHL4中,得到所述O-乙酰-L-高丝氨酸生产菌。
2.如权利要求1所述的O-乙酰-L-高丝氨酸生产菌,其特征在于所述源自Bacillus cereus的基因metA核苷酸序列如SEQ ID No.1所示。
3.构建权利要求1所述的O-乙酰-L-高丝氨酸生产菌的方法,其特征在于所述方法如下:
(1)运用CRISPR-Cas9基因编辑技术,将底盘菌E. coli W3110基因组中的metA、metB、 thrB和metJ基因敲除,得到重组菌株W3110 △metA△metB△thrB△metJ,记为OAHL4;
(2)将核苷酸序列如SEQ ID No.1所示的外源基因metA整合入质粒pACYCDuet-1中,得到质粒pACYCDuet-metA;将质粒pACYCDuet-metA导入步骤(1)所述重组菌OAHL4中,得到所述O-乙酰-L-高丝氨酸生产菌。
4.权利要求1所述O-乙酰-L-高丝氨酸生产菌在微生物发酵制备O-乙酰-L-高丝氨酸中的应用。
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O-乙酰-L-高丝氨酸生产菌接种至发酵培养基中,于28~32 ℃、100~500 rpm条件下进行发酵培养36~48h,发酵结束后取发酵液上清分离纯化得到O-乙酰-L-高丝氨酸;所述发酵培养基组成如下:葡萄糖10~30g/L、硫酸铵10~20g/L、酵母浸粉1~5g/L、KH2PO4 1~5g/L、MgSO4 0.1~2.0g/L、CaCO3 10~20g/L、微量金属盐溶液0.5~5mL/L,pH 6.5~7.0,溶剂为去离子水;微量金属盐溶液组成为:10 g/L CuCl2、10 g/L FeSO4·7H2O、1 g/L ZnSO4·7H2O、0.20 g/L CuSO4、0.02 g/LNiCl2·7H2O,溶剂为去离子水。
6.如权利要求5所述的应用,其特征在于所述发酵培养基组成如下:葡萄糖20g/L,硫酸铵16g/L,酵母粉2g/L,KH2PO4 1g/L,MgSO4 0.2g/L,CaCO3 15g/L,微量金属盐溶液 1mL/L,溶剂为去离子水。
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