CN113278644B - 一种丰原素高产人工菌株的构建及发酵方法 - Google Patents
一种丰原素高产人工菌株的构建及发酵方法 Download PDFInfo
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Abstract
本发明提出一种丰原素高产菌株的构建及发酵方法;利用CRISPR基因编辑将来自解淀粉芽孢杆菌FZB42的4’‑磷酸泛酰巯基乙胺基转移酶基因sfp和枯草芽孢杆菌168的多效因子基因degQ与强启动子P43串联整合至枯草芽孢杆菌168基因组上,构建菌株B.subtilis ds;利用大肠杆菌BL21的乙酰辅酶A合成酶基因acs、于肠沙门氏菌的乙酰辅酶A羧化酶基因accACD和谷氨酸棒状杆菌的生物素连接酶基因birA串联至表达载体pHT43,并导入丰原素合成菌株B.subtilis ds中,构建了丰原素高产菌株B.subtilis dspabacd;构建好的工程菌株用摇瓶发酵培养,生产丰原素。
Description
技术领域
本发明属于微生物技术领域,涉及通过将4’-磷酸泛酰巯基乙胺基转移酶基因sfp调控因子基因degQ串联整合至枯草芽孢杆菌168的基因组上,以及过表达酰辅酶A合成酶基因acs、生物素连接酶基因birA和乙酰辅酶A羧化酶基因accACD,构建能够高产丰原素菌株的方法。
背景技术
丰原素(fengycin)是一种由芽孢杆菌属通过非核糖体肽合成途径合成的,由一条含16个至18个碳原子的β-羟基脂肪酸链和十个氨基酸残基的多肽构成,丰原素对丝状真菌会产生良好的抑制作用,迄今为止,丰原素对真菌阻抑作用机制尚无定论,目前被学术界较为接受的理论是丰原素与细胞的磷脂双分子层发生相互作用,穿透磷脂双分子层,使得细胞膜脂质层的有序性遭到破坏,杀死细胞。在食品、医药、农林业等领域具有很好的应用下效果和发展前景。但是,目前国内的丰原素产量仍处于较低水平,这严重限制了对丰原素的应用发展,因此开发能够提高丰原素产量的方法迫在眉睫。于此同时,木质纤维素木质纤维素是地球上最丰富的可再生资源之一,全球每年可产生约100亿吨木质纤维素,主要由纤维素(35%-50%)、半纤维素(20%-35%)和木质素(5%-30%)组成;纤维素在强酸、或强碱、或酶作用下,水解主要生成葡萄糖;半纤维素,水解后主要生成木糖,因此木糖是丰富的可再生资源。
近年来,随着对丰原素的深入研究,研究者发现,使用现代分子生物学技术对原始菌细胞进行定向的改造,打通目的产物合成限速途径、删减副代谢途径通量、增强生产菌株对代谢产物和代谢废物的容忍性能和对整个代谢网络的改造优化已成为提高丰原素产量的有效方法。例如Zhu等人将丰原素下游合成片段基因导入贝莱斯芽孢杆菌HN-Q-8中并对其进行发酵,发酵产物对马铃薯黑痣病有明显的抑制效果(CN202010339288.0)。Lu等人通过选育得到一株丰原素生产菌株LPB-18N,并通过同源重组技术构建了一种敲除FenSr3基因的重组解淀粉芽孢杆菌,使得丰原素产量提升为327.598mg/L,为原始野生菌株产量的1.72倍(CN201911402559.6)。
枯草芽孢杆菌168具有丰原素合成完整基因簇和高效蛋白合成分泌能力,可以作为丰原素生产的优秀菌株,但是由于其中4’-磷酸泛酰巯基乙胺基转移酶基因(sfp)发生了突变而不能完成非核糖体肽合成酶的活化,不具备催化活性,不能合成丰原素。因此,使枯草芽孢杆菌168具备丰原素合成能力具有重大意义。
脂肪酸是脂肽类抗生素合成的重要前体物质,Yu等人将生物素羧化酶基因转化至原始菌株构建而成,其过表达生物羧化酶,强化了表面活性素分子结构中脂肪酸的合成,使表面活性素产量最高可以提高47%(CN201810865295.7)。但脂肪酸对丰原素合成的影响尚未有研究,因此,增大胞内脂肪酸的合成对提升丰原素的产量有重要意义。
本发明以枯草芽孢杆菌168为研究对象,利用CRISPR技术将4’-磷酸泛酰巯基乙胺基转移酶基因sfp和调控因子基因degQ串联整合至枯草芽孢杆菌168的基因组上,构建了丰原素合成菌株。但是此时丰原素的产量仍旧很低,利用基因工程技术将来自于乙酰辅酶A合成酶基因acs、生物素连接酶基因birA和乙酰辅酶A羧化酶基因accACD串联至表达载体pHT43中并导入具备丰原素合成能力的枯草芽孢杆菌168中,构建了丰原素高产菌株。
发明内容
本发明的目的首先是通过CRISPER基因编辑技术将由4’-磷酸泛酰巯基乙胺基转移酶基因sfp、强启动子P43以及调控因子基因degQ串联整合至枯草芽孢杆菌168的基因组上,构建了丰原素合成菌株B.subtilis ds。为提高丰原素的产量,在丰原素合成菌株中过表达乙酰辅酶A合成酶基因acs、乙酰辅酶A羧化酶基因accACD和生物素连接酶基因birA,促进胞内脂肪酸的合成,为丰原素的合成提供更多的前体物质,以此增大丰原素的产量,构建了一株丰原素高产菌株B.subtilis dspabacd。该菌株的构建对提高丰原素产量具有实际的应用价值。
本发明的目的通过以下技术方案实现:
一种丰原素高产菌株的构建方法;其特征在于利用CRISPR基因编辑技术将来自解淀粉芽孢杆菌FZB42(购自于Prof.Dr.Rainer Borriss,Nordreet UG)的4’-磷酸泛酰巯基乙胺基转移酶基因sfp和来自枯草芽孢杆菌168的多效因子基因degQ与强启动子P43串联整合至枯草芽孢杆菌168的基因组上,构建了一株丰原素合成菌株B.subtilis ds。在丰原素合成菌株B.subtilis ds的基础上为提高丰原素的产量,利用基因工程技术将来自大肠杆菌BL21的乙酰辅酶A合成酶基因acs、来自于肠沙门氏菌的乙酰辅酶A羧化酶基因accACD和来自于谷氨酸棒状杆菌的生物素连接酶基因birA串联至表达载体pHT43,并将其导入丰原素合成菌株B.subtilis ds中,构建了一株丰原素高产菌株B.subtilis dspabacd。
丰原素高产菌株的构建发酵包括如下步骤:
1)根据sfp基因的已知核苷酸序列设计PCR引物,以解淀粉芽孢杆菌FZB42基因组为模板扩增sfp基因;
2)根据degQ基因的已知核苷酸序列设计PCR引物,以枯草芽孢杆菌168基因组为模板扩增degQ基因;
3)根据P43启动子的已知核苷酸序列设计PCR引物,以枯草芽孢杆菌168基因组为模板扩增P43启动子;
4)将sfp基因、degQ基因、P43启动子通过融合PCR串联成一整个基因片段;
5)将4)中的融合片段连入CRISPR质粒pJOE8999质粒,获得携带sfp基因、degQ基因、P43启动子的重组质粒pJOEDSP;
6)根据acs基因的已知核苷酸序列、birA基因的已知核苷酸序列(和accACD基因的已知核苷酸序列,合成acs、birA、accACD和P43串联的融合片段,并连入表达载体pHT43,获得携带birA基因、acs基因、accACD基因和P43启动子的重组质粒pHTABPC;
7)将5)中构建好的重组质粒pJOEDSP通过化转的方法整合至枯草芽孢杆菌168的基因组上,构建sfp和degQ双基因整合至枯草芽孢杆菌168的人工菌株B.subtilis ds;
8)将6)中构建好的重组质粒pHTABPC通过化转的方法导入人工菌株B.subtilisds细胞内,构建birA基因、acs基因、accACD基因过表达菌株B.subtilis dspabacd;
9)培养7)和8)中构建好的工程菌株用摇瓶发酵培养,生产丰原素。
所述的sfp基因序列为SEQ ID NO:1。
所述的degQ基因序列为SEQ ID NO:2。
所述的P43启动子的序列为SEQ ID NO:3。
所述的acs基因序列为SEQ ID NO:4。
所述的birA基因序列为SEQ ID NO:5。
所述的accACD基因序列为SEQ ID NO:6。
所述的发酵培养液为:木糖20g/L,水溶性黄豆饼粉21.9g/L,NaNO3 3.1g/L,MnSO4·H2O0.2g/L,pH 7.5。
利用本发明的方法构建的丰原素合成菌株B.subtilis ds,丰原素的产量为37.92mg/L;丰原素高产菌株B.subtilis dspabacd丰原素的产量为130.11mg/L,提高了343%。本发明提出的方法为提高丰原素产量提供了一种新的策略,在丰原素的工业化生产中具有实际的应用价值和潜在的商业价值。
附图说明
图1:P43扩增基因的电泳结果;
图2:P43+sfp融合片段的电泳结果;
图3:P43+sfp+degQ融合片段的电泳结果;
图4:构建P43+sfp+degQ串联重组质粒pJOEDSP的流程图;
图5:构建acs、birA和accACD三基因过表达质粒pHTABPC的流程图;
图6:B.subtilis ds及B.subtilis dspabacd的丰原素产量对比图。
具体实施方式
为了使本发明的目的、技术方案和效果更加清晰,下面结合附图和具体的实施例对本发明进行详细说明,下述实施例是说明性的,不是限定性的,本领域的专业人员按照本发明的精神可以对其进行改进和变化,所述的这些改进和变化都应当视为在本发明的范围内,本发明的范围和实质由权力要求来限定。
实施例一:丰原素合成菌株B.subtilis ds的构建
(1)sfp基因扩增引物的设计与合成。根据sfp基因的核酸序列(如SEQ ID NO:1所示),设计扩增引物如下(下划直线表示SalⅠ酶切位点,下划波浪线表示BglⅠⅠ酶切位点),引物由擎科生物科技有限公司进行合成。
SFP-F:AAGGCCAACGAGGCCCAAAAGAAGAACGGACACAGCGGTTC
SFP-R:
(2)解淀粉芽孢杆菌FZB42基因组的提取。取3-5mL于LB液体培养基中37℃,220rpm/min培养12-16h的菌液,于4℃、12000rpm离心30s,收集沉淀。沉淀中加入500μL的细胞裂解液,上下颠倒晃动7-8次,于37℃金属浴保持30min。向上述裂解物中加入264μL的5MNaCl溶液,上下颠倒晃动7-8次,于12000rpm离心10min,将上清液转移至新取得干净无菌离心管中。加入500μL的饱和苯酚/氯仿,轻轻用移液枪不断抽吸,充分混匀,12000rpm离心30min,上清液转移至新取的干净无菌离心管中,重复上述步骤一次。加500μL氯仿,上下颠倒晃动7-8次,12000rpm离心30min,上清液转移至干净无菌的离心管中。加入4℃预冷的200μL无水乙醇,放置于-20℃条件下进行沉淀30min,然后以12000rpm离心10min,倒掉液体,保收集白色丝状沉淀。加入4℃预冷的500μL 70%乙醇洗涤,重复洗涤两次,室温干燥后,用60μL无菌去离子水溶解沉淀,即完成提取基因组过程,存放于冰箱备用。
(3)sfp基因的扩增。以上述提取的解淀粉芽孢杆菌FZB42基因组为模板,以上述的SFP-F和SFP-R为引物扩增sfp基因。PCR反应体系为:总体积50μL,无菌水17μL,2×PhantaMax Buffer 25μL,dNTP Mix 1μL,上游引物2μL,下游引物2μL,Phanta Max SuperFidelity DNA Polymerase 1μL,模板2μL;PCR反应程序为:95℃3min,95℃15s,72℃60s/kb,72℃5min,4℃保温,循环数:30。
(4)degQ基因扩增引物的设计与合成。根据degQ基因的核酸序列(如SEQ ID NO:2所示),设计扩增引物如下(下划直线表示BglⅠⅠ酶切位点,下划波浪线表示EcoRⅠ酶切位点),引物由擎科生物科技有限公司进行合成。
DEGQ-F:
GAAGATCTTTTGCAGACGGAGGATCTAGAGCGGCTTCCTTGTAGAGCTCAGCATTATTG
DEGQ-R:
(5)枯草芽孢杆菌168基因组的提取。取3-5mL于LB液体培养基中37℃,220rpm/min培养12-16h的菌液,于4℃、12000rpm离心30s,收集沉淀。沉淀中加入500μL的细胞裂解液,上下颠倒晃动7-8次,于37℃金属浴保持30min。向上述裂解物中加入264μL的5MNaCl溶液,上下颠倒晃动7-8次,于12000rpm离心30min,将上清液转移至新取得干净无菌离心管中。加入500μL的饱和苯酚/氯仿,轻轻用移液枪不断抽吸,充分混匀,12000rpm离心30min,上清液转移至新取的干净无菌离心管中,重复上述步骤一次。加500μL氯仿,上下颠倒晃动7-8次,12000rpm离心30min,上清液转移至干净无菌的离心管中。加入4℃预冷的200μL无水乙醇,放置于-20℃条件下进行沉淀30min,然后以12000rpm离心10min,倒掉液体,保收集白色丝状沉淀。加入4℃预冷的500μL 70%乙醇洗涤,重复洗涤两次,室温干燥后,用60μL无菌去离子水溶解沉淀,即完成提取基因组过程,存放于冰箱备用。
(6)dsgQ基因的扩增。以上述提取的枯草芽孢杆菌168基因组为模板,以上述的DEGQ-F和DEGQ-R为引物扩增dsgQ基因。PCR反应体系为:总体积50μL,无菌水17μL,2×Phanta Max Buffer 25μL,dNTP Mix 1μL,上游引物2μL,下游引物2μL,Phanta Max SuperFidelity DNA Polymerase 1μL,模板2μL;PCR反应程序为:95℃3min,95℃15s,72℃60s/kb,72℃5min,4℃保温,循环数:30。
(7)P43启动子扩增引物的设计与合成。根据P43启动子的核酸序列(如SEQ ID NO:3所示),设计扩增引物如下(下划波浪线表示SalⅠ酶切位点),引物由擎科生物科技有限公司进行合成。
P43-F:CCCAAGCTTGCGGCTTCCTTGTAGAGCTCAGC
P43-R:
(8)P43启动子的扩增。以上述提取的枯草芽孢杆菌168基因组为模板,以上述的P43-F和P43-R为引物扩增P43启动子。PCR反应体系为:总体积50μL,无菌水17μL,2×PhantaMax Buffer 25μL,dNTP Mix 1μL,上游引物2μL,下游引物2μL,Phanta Max SuperFidelity DNA Polymerase 1μL,模板2μL;PCR反应程序为:95℃3min,95℃15s,72℃60s/kb,72℃5min,4℃保温,循环数:30,扩增结果如图1所示。
(9)P43和sfp的融合。以上述P43和sfp为模板,以上述的SFP-F和P43-R为引物进行P43和sfp的融合。PCR反应体系为:总体积50μL,无菌水17μL,2×Phanta Max Buffer 25μL,dNTP Mix 1μL,上游引物2μL,下游引物2μL,Phanta Max Super Fidelity DNA Polymerase1μL,模板2μL;PCR反应程序为:95℃3min,95℃15s,72℃60s/kb,72℃5min,4℃保温,循环数:30,P43和sfp的融合结果如图2所示。
(10)P43和sfp的融合片段与dsgQ的融合。以(9)中融合产物和dsgQ为模板,以上述的DEGQ-F和P43-R为引物进行融合。PCR反应体系为:总体积50μL,无菌水17μL,2×PhantaMax Buffer 25μL,dNTP Mix 1μL,上游引物2μL,下游引物2μL,Phanta Max SuperFidelity DNA Polymerase 1μL,模板2μL;PCR反应程序为:95℃3min,95℃15s,72℃60s/kb,72℃5min,4℃保温,循环数:30,P43和sfp的融合片段与dsgQ的融合结果如图3所示。
(11)sfp和degQ双基因连接在pJOE8999质粒的重组质粒pJOEDSP的构建及筛选。构建流程如图4所示,将(10)中的融合片段用限制性内切酶NdeⅠ和XbaⅠ进行酶切反应,同时将质粒pJOE8999也进行NdeⅠ和XbaⅠ双酶切反应,用T4连接酶将含酶切末端的融合片段与含相同酶切末端的质粒pJOE8999中过夜进行酶连反应。将连接产物转化进入大肠DH5α感受态中,涂布于含50μg/mL卡那霉素和0.2%甘露醇的LB固体培养基(LB固体培养基配方:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L,琼脂20g/L)中,37℃培养16-18小时,挑取长出来的单菌落进行菌落PCR验证,验证片段与理论片段大小相符合,即筛选出正确的双基因载体pJOEDSP。对正确得单菌落进行液体扩大培养,提取质粒送擎科生物科技有限公司进行测序验证。
(12)将正确的重组质粒pJOEDSP转化入枯草芽孢杆菌168中,于含有筛选压力(卡那霉素和0.2%甘露醇)的固体平板上进行固体平板培养,30℃倒扣培养11-17h;长出单菌落后,进行菌落PCR实验,筛选正确的单菌落,得到初步确定整合的转化子。将初步确定整合的转化子转移至50℃继续培养,采用基因组验证引物和pJOE8999质粒通用引物进行菌落PCR得到整合成功并且pJOEDSP质粒丢失的转化子。继续于50℃培养得到的转化子,对其进行纯化和完全丢失pJOEDSP质粒,再次PCR后挑取正确的转化子,最终获得B.subtilis ds菌株。
实施例二:丰原素高产菌株B.subtilis dspabacd的构建
(1)依据启动子P43的核酸序列(如SEQ ID NO:3所示)、乙酰辅酶A合成酶基因acs的核酸序列(如SEQ ID NO:4所示)、乙酰辅酶A羧化酶基因accACD的核酸序列(如SEQ IDNO:6所示)和来生物素连接酶基因birA的核酸序列(如SEQ ID NO:5所示),委托擎科生物科技有限公司合成acs、birA、accACD和P43串联的融合片段,并连入表达载体pHT43,构建流程如图5所示,获得携带birA基因、acs基因、accACD基因和P43启动子的重组表达质粒pHTABPC。
(2)将表达质粒pHTABPC通过化转的方式导入B.subtilis ds中,涂布于氯霉素筛选压力平板进行固体平板培养,37℃倒扣静置培养13-17h,挑选单菌落进行验证,筛选出正确的接合子,提取质粒进行验证,最终获得B.subtilis dspabacd菌株。
实施例三:B.subtilis ds和B.subtilis dspabacd的培养及发酵
(1)培养基成分:
种子培养基:胰蛋白胨10g/L,酵母浸粉5g/L,NaCl 10g/L,pH 7.0;
发酵培养基:木糖20g/L,水溶性黄豆饼粉21.9g/L,NaNO3 3.1g/L,MnSO4·H2O0.2g/L,pH 7.5。
(2)B.subtilis ds和B.subtilis dspabacd的培养及发酵。将本发明的丰原素合成菌株B.subtilis ds和丰原素高产菌株B.subtilis dspabacd分别进行固体平板培养,37℃倒扣静置培养13-17h,取单菌落,继续进行固体平板培养上,37℃倒扣静置培养24h,取单菌落,于50mL装液量的液体培养基中,进行种子培养,37℃、220rpm/min培养12h,得到种子液,以体积比为5%的接种量接种至发酵培养基中,37℃、220rpm/min进行发酵,48h时取30mL发酵液于灭菌干燥的50mL专用离心管中,9000rpm离心30min,收集上清液,6M HCl调节pH至2,4℃静置24h,待完全沉淀后,9000rpm离心34min,收集沉淀,用3mL甲醇溶解沉淀,调pH至7.0,4℃条件下静置萃取12h,0.22μm油膜过滤后用于液相色谱的产量分析;液相色谱仪1100(Agilent,USA),色谱柱类型:XDB C-18反相柱(250mm×4.6mm,5μm),流动相:V(乙腈):V(水):V(三氟乙酸)=50:50:0.5,0.8mL/min,30℃,210nm条件进行检测,样品进样20μL。发酵结果如图6所示,发酵产量的检测结果显示利用本发明提出的方法能够使B.subtilis ds具备生产丰原素的能力产量为37.92mg/L,实现了丰原素从0的合成;B.subtilis dspabacd丰原素的产量为130.11mg/L,较B.subtilis ds提高了343%。
本发明提出的方法为提高丰原素产量提供了一种新的策略,在丰原素的工业化生产中具有实际的应用价值和潜在的商业价值。
本发明公开和提出的技术方案,本领域技术人员可通过借鉴本文内容,适当改变条件路线等环节实现,尽管本发明的方法和制备技术已通过较佳实施例子进行了描述,相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和技术路线进行改动或重新组合,来实现最终的制备技术。特别需要指出的是,所有相类似的替换和改动对本领域技术人员来说是显而易见的,他们都被视为包括在本发明精神、范围和内容中。
序列表
<110> 天津大学
<120> 一种丰原素高产人工菌株的构建及发酵方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 325
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggaaaaga aacttgaaga agtaaaacaa ttgttattcc gactcgaact tgatattaaa 60
gaaacgacag attcattacg aaacattaac aaaagcattg atcaactcga taaatacaat 120
tatgcaatga aaatttcgtg aaaaagactt ggaaacaagt cttttttttc gttctaccga 180
tacaataaat ggataaagta ttatatgatt gttaaaaaac gaaaaacctg ctgtccttta 240
aatgtcccat ttagtaaaat ggaatgggag gggggaagtc gttattgagc agatatgttt 300
agattctgtc cggatcaagg agaaa 325
<210> 2
<211> 675
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaaaattt acggagtata tatggaccgc ccgctttctg caggggaaga ggatcggatg 60
atggcggccg tgtccgccga aaagcgggaa aaatgccggc gcttttacca taaggaggat 120
gctcaccgca ccttgatcgg cgacatgctg atccgcaccg ctgcggcgaa ggcttatgga 180
cttgatccgg ccgggatttc attcggcgtc caggaatacg gaaagccgta catccccgcg 240
cttccggaca tgcactttaa catttcccac tccgggcgct ggatcgtgtg cgccgttgat 300
tcaaaaccga tcggcattga tattgaaaaa atgaagcccg gcacaattga tatcgccaaa 360
cggttttttt cgccgacgga atacagtgat ctgcaagcga aacaccccga tcagcagacc 420
gattattttt accacctgtg gtcgatgaaa gaaagcttta tcaagcaggc cggaaaaggg 480
ctttccctgc cgcttgattc attcagcgtc cgccttaaag acgacggcca tgtgtccatt 540
gagctcccgg acggacatga accttgtttc atccgcacat atgaggcgga cgaggagtat 600
aagctggccg tttgtgcggc gcatcccgat ttttgtgacg ggattgagat gaaaacgtat 660
gaagagctgt tataa 675
<210> 3
<211> 426
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgtcgacgtg catgcaggcc ggggcatatg ggaaacagcg cggacggagc ggaatttcca 60
atttcatgcc gcagccgcct gcgctgttct catttgcggc ttccttgtag agctcagcat 120
tattgagtgg atgattatat tccttttgat aggtggtatg ttttcgcttg aacttttaaa 180
tacagccatt gaacatacgg ttgatttaat aactgacaaa catcaccctc ttgctaaagc 240
ggccaaggac gctgccgccg gggctgtttg cgtttttacc gtgatttcgt gtatcattgg 300
tttacttatt tttttgccaa agctgtaatg gctgaaaatt cttacattta ttttacattt 360
ttagaaatgg gcgtgaaaaa aagcgcgcga ttatgtaaaa tataaagtga tagcggtacc 420
attata 426
<210> 4
<211> 1699
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
agaaaacggc gatcgtaccg ccatcatctg ggaaggcgac gacgccagcc agagcaaaca 60
tatcagctat aaagagctgc accgcgacgt ctgccgcttc gccaataccc tgctcgagct 120
gggcattaaa aaaggtgatg tggtggcgat ttatatgccg atggtgccgg aagccgcggt 180
tgcgatgctg gcctgcgccc gcattggcgc ggtgcattcg gtgattttcg gcggcttctc 240
gccggaagcc gttgccgggc gcattattga ttccaactca cgactggtga tcacttccga 300
cgaaggtgtg cgtgccgggc gcagtattcc gctgaagaaa aacgttgatg acgcgctgaa 360
aaacccgaac gtcaccagcg tagagcatgt ggtggtactg aagcgtactg gcgggaaaat 420
tgactggcag gaagggcgcg acctgtggtg gcacgacctg gttgagcaag cgagcgatca 480
gcaccaggcg gaagagatga acgccgaaga tccgctgttt attctctaca cctccggttc 540
taccggtaag ccaaaaggtg tgctgcatac taccggcggt tatctggtgt acgcggcgct 600
gacctttaaa tatgtctttg attatcatcc gggtgatatc tactggtgca ccgccgatgt 660
gggctgggtg accggacaca gttacttgct gtacggcccg ctggcctgcg gtgcgaccac 720
gctgatgttt gaaggcgtac ccaactggcc gacgcctgcc cgtatggcgc aggtggtgga 780
caagcatcag gtcaatattc tctataccgc acccacggcg atccgcgcgc tgatggcgga 840
aggcgataaa gcgatcgaag gcaccgaccg ttcgtcgctg cgcattctcg gttccgtggg 900
cgagccaatt aacccggaag cgtgggagtg gtactggaaa aaaatcggca acgagaaatg 960
tccggtggtc gatacctggt ggcagaccga aaccggcggt ttcatgatca ccccgctgcc 1020
tggcgctacc gagctgaaag ccggttcggc aacacgtccg ttcttcggcg tgcaaccggc 1080
gctggtcgat aacgaaggta acccgctgga gggggccacc gaaggtagcc tggtaatcac 1140
cgactcctgg ccgggtcagg cgcgtacgct gtttggcgat cacgaacgtt ttgaacagac 1200
ctacttctcc accttcaaaa atatgtattt cagcggcgac ggcgcgcgtc gcgatgaaga 1260
tggctattac tggataaccg ggcgtgtgga cgacgtgctg aacgtctccg gtcaccgtct 1320
ggggacggca gagattgagt cggcgctggt ggcgcatccg aagattgccg aagccgccgt 1380
agtaggtatt ccgcacaata ttaaaggtca ggcgatctac gcctacgtca cgcttaatca 1440
cggggaggaa ccgtcaccag aactgtacgc agaagtccgc aactgggtgc gtaaagagat 1500
tggcccgctg gcgacgccag acgtgctgca ctggaccgac tccctgccta aaacccgctc 1560
cggcaaaatt atgcgccgta ttctgcgcaa aattgcggcg ggcgatacca gcaacctggg 1620
cgatacctcg acgcttgccg atcctggcgt agtcgagaag ctgcttgaag agaagcaggc 1680
tatcgcgatg ccatcgtaa 1699
<210> 5
<211> 867
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttgggcgtgt cgcccttaaa gcgcgctttt cgacgcgacc ccactacatt ggcttccatg 60
aacgttgaca tttcacgatc cagagagccg ctaaacgttg agctcctgaa ggaaaaattg 120
ctccaaaacg gtgactttgg ccaggtcatt tacgaaaaag tgacaggctc cactaatgct 180
gacttgctgg cacttgcagg ttctggcgct ccaaactgga cggtgaaaac tgtcgagttt 240
caagatcatg cgcgtgggcg actcggccgc ccgtggtctg cccctgaggg ttcccaaaca 300
atcgtgtctg tgctcgttca actatctatt gatcaagtgg accggattgg cactattcca 360
ctcgcggcgg gactcgctgt catggatgcg ttgaatgacc tcggtgtgga aggtgccgga 420
ctgaaatggc ccaacgatgt tcaaatccac ggcaagaaac tctgcggcat cctggtggaa 480
gccaccggct ttgattccac cccaacagtt gtcatcggtt ggggcactaa tatcagcctg 540
actaaagagg agcttcctgt tcctcatgca acttccctcg cattggaagg tgttgaagtc 600
gacagaacca cattccttat taatatgctc acacatctgc atactcgact ggaccagtgg 660
cagggtccaa gtgtggattg gctcgatgat taccgtgcgg tatgttccag tattggccaa 720
gatgttcgag tgcttctacc tggggataaa gaactcttag gtgaagcgat cggtgtcgcg 780
actggcggag aaattcgtgt tcgcgatgct tcgggcaccg ttcacaccct caacgccggt 840
gaaattacgc accttcgcct gcagtaa 867
<210> 6
<211> 3431
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tggcgcaacg tggcgtgtct ctccgagcga tcgtttactt aacgatctgc gtggcctcat 60
tggttcggag caggtggaac tggagtttga ctaatacagg aatactatga gtctgaattt 120
ccttgatttt gaacagccga tagcagagct ggaagcgaaa atcgattctc tgactgcggt 180
aagccgccag gatgagaaac tggatattaa tatcgatgaa gaagtccatc gcctgcgcga 240
aaaaagcgta gaactgacgc gcaaaatctt cgccgatctc ggcgcatggc aggtagccca 300
actggcgcgc catccgcagc gtccgtacac cctggattat gtccgtctgg cgtttgatga 360
gtttgatgag ctggcgggcg atcgcgccta tgctgatgac aaagccatcg tcggcggtat 420
cgcgcgtctg gaagggcgac cggtgatgat cattggtcat cagaaaggtc gtgaaaccaa 480
agaaaaaatt cgtcgtaact ttggcatgcc agcgccggaa ggctaccgca aagcgctgcg 540
cctgatggaa atggctgaac gcttcaacat gccgattatc accttcatcg acaccccggg 600
tgcataccct ggcgtgggcg cggaagagcg tggtcagtct gaagccatcg cccgcaacct 660
gcgtgagatg tctcgtctga acgtaccggt catctgcacc gtgattggcg aaggcggctc 720
cggcggcgca ttggcgattg gcgtgggtga taaagtgaat atgctgcaat acagcaccta 780
ttccgttatc tccccggaag gttgcgcctc tattttgtgg aagagcgccg acaaagcacc 840
attggctgct gaagcgatgg gcatcatcgc gccgcgtctg aaagagctga agctaatcga 900
ttccatcatt ccggagccgc tgggcggcgc gcaccgtaat ccggaagcga tggcggcatc 960
gctaaaagcg cagctgctgg aagatttagc cgatctcgat gtgttaagca ccgatgattt 1020
aaaaaaccgt cgttaccagc gtttgatgag ctacggttac gcctaagagg cgaacatgtt 1080
ggataaaatt gtcatcgcca accgcggcga gatcgcacta cgtattcttc gagcctgtaa 1140
agaactcggc atcaagaccg tcgctgtgca ctcaagcgcg gatcgcgatt taaaacacgt 1200
attgctggcg gatgagacgg tctgtattgg tccggcaccg tccgtaaaaa gttatctgaa 1260
catcccggct atcattagcg ccgctgaaat caccggcgcg gtggcaatcc acccgggtta 1320
cggcttcctt tctgagaacg ccaattttgc cgagcaggtt gaacgctccg gctttatctt 1380
tatcggcccg aaagcggaca ccatccgcct gatgggcgat aaagtgtccg cgattaccgc 1440
gatgaaaaaa gcgggcgtgc cgaccgtacc aggatctgac ggcccgctgg gcgacgatat 1500
gaatgcgaac cgcgctcatg ccaaacgtat tggctatccg gtgatcatca aagcgtccgg 1560
cggcggcggc ggccgcggta tgcgcgtagt tcgtagcgat gctgaactgg cgcagtccat 1620
ctccatgacc aaagcggaag cgaaagcagc tttcagcaac gacatggtat acatggaaaa 1680
atacctggaa aatccacgcc acatcgaaat tcaggtgctg gcggacggcc agggcaacgc 1740
tatctatctg gcggaacgcg actgttccat gcagcgtcgc caccagaaag tggttgaaga 1800
agccccggcg ccaggcatta cgccggaact gcgtcgctat atcggcgaac gctgcgcgaa 1860
agcgtgcgta gacatcggct atcgcggcgc agggacgttc gaattcctgt tcgaaaacgg 1920
cgagttctat ttcatcgaaa tgaacacccg tattcaggtt gaacacccgg tgactgaaat 1980
gattaccggc gtcgatttga tcaaagagca gttgcgcatc gcggcgggtc agccgctgtc 2040
gatcacacag gacgaagttg tcgttcgagg ccatgcggta gaatgccgta tcaatgccga 2100
agatccgaac accttcctgc caagcccagg caaaatcacg cgcttccatg cgcctggcgg 2160
ctttggcgtt cgttgggaat cccatatcta cgcgggctac acggtgccgc cgtactatga 2220
ttccatgatc ggcaaactca tctgctacgg tgaaaaccgc gacgtggcga ttgcccgtat 2280
gaaaaatgcc ctgcaggaac tgattatcga tggtatcaaa accaatatcg atctgcagac 2340
ccgcatcatg aatgacgagc acttccagca cggtggaacc aacatccact atctggagaa 2400
aaaactcggt cttcaggaaa agtaaatgtg caacattcat ggtctgttgg gggcaaaaat 2460
ggcattatgc gccccttata ataaagctga ctaaggttca ggcagaaagg tcaccaatga 2520
gctggattga acgaattaaa agcaacatta ctcccacccg caaggctagc attcctgaag 2580
gggtgtggac caagtgtgat agctgcggtc aggttttata ccgcgctgag ctggaacgta 2640
atcttgaggt ctgtcctaag tgtgaccatc atatgcgcat gtcggcgcgc aatcgcctgc 2700
atagcctgtt agatgaaggt tcccttgtgg agttgggtag cgagctggag ccaaaagacg 2760
tgctcaagtt ccgcgactcc aaaaaatata aagacagact ggcgtccgcg cagaaagaaa 2820
ccggcgagaa agacgcgctg gtggtcatga aagggacgct tcacggtatg ccggttgtcg 2880
ccgccgcgtt tgaattcgcg ttcatgggcg gctcaatggg gtctgtcgtt ggcgcacgct 2940
tcgttcgcgc cgttgaacag gcgctggaag acaactgtcc gttagtgtgt ttctctgctt 3000
ccggcggcgc gcgtatgcaa gaagcgctga tgtcgctgat gcagatggcg aaaacctcgg 3060
cggcgctggc taaaatgcag gaacgtggtc tgccctacat ttcggtattg accgatccga 3120
caatgggcgg cgtttccgcc agttttgcga tgctgggcga tctcaacatc gccgagccaa 3180
aagctctgat tggcttcgcc ggcccgcgcg ttatcgaaca gaccgttcgc gaaaaactgc 3240
cgccaggatt ccagcgcagt gagttcctga tcgaaaaagg cgctattgat atgatagtcc 3300
gccgcccgga aatgcgcctg aagctggcga gcattctggc gaagctgatg aatctgcccg 3360
cgccgaaccc ggacgccccg cgtgaaggcg tggtggtgcc tccggcgccg gatcaggagt 3420
ctgaggcctg a 3431
Claims (9)
1.一种丰原素高产菌株的构建及发酵方法;其特征在于利用CRISPR基因编辑技术将来自解淀粉芽孢杆菌FZB42的4’-磷酸泛酰巯基乙胺基转移酶基因sfp和来自枯草芽孢杆菌168的多效因子基因degQ与强启动子P43串联整合至枯草芽孢杆菌168的基因组上,构建了一株丰原素合成菌株B.subtilis ds;利用基因工程技术将来自大肠杆菌BL21的乙酰辅酶A合成酶基因acs、来自于肠沙门氏菌的乙酰辅酶A羧化酶基因accACD和来自于谷氨酸棒状杆菌的生物素连接酶基因birA串联至表达载体pHT43,并将其导入丰原素合成菌株B.subtilis ds中,构建了一株丰原素高产菌株B.subtilis dspabacd;构建好的工程菌株用摇瓶发酵培养,生产丰原素。
2.如权利要求1所述的方法,其特征是包括如下步骤:
1)根据sfp基因的已知核苷酸序列设计PCR引物,以解淀粉芽孢杆菌FZB42基因组为模板扩增sfp基因;
2)根据degQ基因的已知核苷酸序列设计PCR引物,以枯草芽孢杆菌168基因组为模板扩增degQ基因;
3)根据P43启动子的已知核苷酸序列设计PCR引物,以枯草芽孢杆菌168基因组为模板扩增P43启动子;
4)将sfp基因、degQ基因、P43启动子通过融合PCR串联成一整个基因片段;
5)将4)中的融合片段连入CRISPR质粒pJOE8999质粒,获得携带sfp基因、degQ基因、P43启动子的重组质粒pJOEDSP;
6)根据acs基因的已知核苷酸序列、birA基因的已知核苷酸序列和accACD基因的已知核苷酸序列,合成acs、birA、accACD和P43串联的融合片段,并连入表达载体pHT43,获得携带birA基因、acs基因、accACD基因和P43启动子的重组质粒pHTABPC;
7)将5)中构建好的重组质粒pJOEDSP通过化转的方法整合至枯草芽孢杆菌168的基因组上,构建sfp和degQ双基因整合至枯草芽孢杆菌168的人工菌株B.subtilis ds;
8)将6)中构建好的重组质粒pHTABPC通过化转的方法导入人工菌株B.subtilis ds细胞内,构建birA基因、acs基因、accACD基因过表达菌株B.subtilis dspabacd;
9)培养7)和8)中构建好的工程菌株用摇瓶发酵培养,生产丰原素。
3.如权利要求2所述的方法,其特征是sfp基因序列为SEQ ID NO:1。
4.如权利要求2所述的方法,其特征是degQ基因序列为SEQ ID NO:2。
5.如权利要求2所述的方法,其特征是P43启动子的序列为SEQ ID NO:3。
6.如权利要求2所述的方法,其特征是acs基因序列为SEQ ID NO:4。
7.如权利要求2所述的方法,其特征是birA基因序列为SEQ ID NO:5。
8.如权利要求2所述的方法,其特征是accACD基因序列为SEQ ID NO:6。
9.如权利要求2所述的方法,其特征是发酵培养液为:木糖20g/L,水溶性黄豆饼粉21.9g/L,NaNO3 3.1g/L,MnSO4·H2O 0.2g/L,pH 7.5。
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CN110951667B (zh) * | 2019-12-30 | 2023-03-24 | 淮阴工学院 | 一株丰原素高产菌株lpb-18n及其选育和应用 |
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