CN117417952B - 一种提高香兰素产量的重组拟无枝酸菌、其构建方法及应用 - Google Patents
一种提高香兰素产量的重组拟无枝酸菌、其构建方法及应用 Download PDFInfo
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Abstract
本发明提供调控蛋白tyrR1基因缺失以及tyrA‑aroF基因的过表达在提高拟无枝酸菌以葡萄糖为底物发酵产香兰素的香兰素产量重点的应用,本发明还提供一种高产香兰素产量的重组拟无枝酸菌,所述重组拟无枝酸菌敲除了拟无枝酸菌的调控蛋白基因tyrR1和丙酮酸激酶基因pyk,过表达拟无枝酸菌内源的分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3‑脱氧‑D‑***庚酮糖酸‑7‑磷酸(DAHP)合成酶基因aroF和磷酸烯醇式丙酮酸合酶基因ppsA。本发明的重组拟无枝酸菌能够以葡萄糖为底物发酵产香兰素,其产量实现4.72g/L,显著高于现有技术。
Description
技术领域
本发明属于基因工程技术领域。更具体地,本发明涉及一种提高香兰素产量的重组拟无枝酸菌,还涉及所述拟无枝酸菌在生产香兰素中的应用。
背景技术
香兰素(3-甲氧基-4-羟基苯甲醛),又称香草醛,是世界上使用最广泛的调味料之一,在食品、药品、化妆品、农业等领域被广泛应用。目前全球对香兰素的需求约为2万吨。目前市场上香兰素的供应有三种,(1)从香荚兰豆中提取的天然香兰素;(2)用化学合成法生产的香兰素;(3)用微生物转化法生产的香兰素。由于植物提取法和化学合成法存在种种不足,致使人们越来越重视微生物转化法生产香兰素,其中,丁香酚、异丁香酚和阿魏酸是该法生产香兰素的主要底物。但是丁香酚、异丁香酚毒性大,阿魏酸价格高,因此,寻求安全廉价的原料来生产香兰素是一个重要的研究方向。
由于葡萄糖价格低廉,原料充足,且安全无毒,是生物合成香兰素的理想原料。Hansen等研究表明,通过代谢工程改造后的酵母菌株,包括裂殖酵母和酿酒酵母分别以葡萄糖为底物,可获得65mg/L和45mg/L的香兰素,然而酵母具有较强的香兰素代谢能力,使得产量降低和副产物的生成。中国发明专利申请CN 106032538A构建了一株代谢工程大肠杆菌,通过转入5个外源基因,经过苯丙烷途径生成香兰素,但其产量低,且香兰素对大肠杆菌毒性大,不利于其积累。发明专利申请CN 114703113A通过在拟无枝酸菌中表达酪氨酸解氨酶基因tal、对香豆酸-3-羟化酶基因sam5和咖啡酸甲基转移酶基因com,并缺失预苯酸脱水酶基因,实现了以葡萄糖为原料生物合成香兰素,摇瓶发酵的香兰素产量为1.25g/L。该专利申请虽然能够从头合成香兰素,但是香兰素产量仍旧较低,难以实现产业化生产。为了提高香兰素的产量,合成香兰素的重要前体物质酪氨酸的通量需要被加强,而合成酪氨酸主要在大肠杆菌中研究较多,目前没有报道有关拟无枝酸菌增强酪氨酸的研究。
发明内容
本发明的目的是克服现有技术方案,通过基因改造提供一种高产香兰素产量的重组拟无枝酸菌。
基于该目的,本发明提供拟无枝酸菌调控蛋白tyrR1基因缺失在提高拟无枝酸菌以葡萄糖为底物发酵产香兰素的香兰素产量重点的应用,所述tyrR1的基因序列如SEQ IDNO:1所示,其编码的蛋白质登录号为WP_020416143.1。
本发明还提供过表达tyrA-aroF基因在提高拟无枝酸菌以葡萄糖为底物发酵产香兰素的香兰素产量重点的应用,所述tyrA是分支酸变位酶/预苯酸脱氢酶双功能酶基因,其核酸序列如SEQ ID NO:2所示,其编码的蛋白质登录号为WP_020419478.1,所述aroF基因是3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因,aroF的基因序列如SEQ ID NO:3所示,其编码的蛋白质登录号为WP_020418838.1。
进一步地,本发明提供一种高产香兰素产量的重组拟无枝酸菌,所述重组拟无枝酸菌敲除了拟无枝酸菌(Amycolatopsis sp.)的调控蛋白基因tyrR1和丙酮酸激酶基因pyk,过表达拟无枝酸菌内源的分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroF和磷酸烯醇式丙酮酸合酶基因ppsA。
在本发明中,基因tyrR1的核苷酸序列如SEQ ID NO:1所示,基因tyrA的核苷酸序列如SEQ ID NO:2所示,基因aroF的核苷酸序列如SEQ ID NO:3所示,基因pyk的核苷酸序列如SEQ ID NO:4所示,基因ppsA的核苷酸序列如SEQ ID NO:5所示。
另一方面,本发明还提供上述重组拟无枝酸菌的构建方法,所述方法包括以下步骤:
(1)构建tyrR1基因缺失的重组拟无枝酸菌
以VA-F2/VA-R2和VA-F3/VA-R3为引物,拟无枝酸菌基因组为模板,PCR扩增tyrR1基因的上下游同源臂,同时将所得的两个tyrR1-up-2500和tyrR1-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-tyrR1-2500;
利用接合转移实验方法,将得到的pKG1132-tyrR1-2500质粒转化到拟无枝酸菌出发菌株中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-145基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因缺失的重组拟无枝酸菌;
(2)构建整合tyrA、aroF基因的重组拟无枝酸菌
以VA-F1/VA-R1为引物,pKC1139-permE为模板,PCR扩增permE片段,将permE片段通过Assembly试剂盒连接到pSET152质粒的PvuI/EcoRV位点,构建得到质粒pSET152-permE;以VA-F8/VA-R8为引物,拟无枝酸菌基因组为模板,PCR扩增tyrA基因,将tyrA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-tyrA;以VA-F10/VA-R10为引物,拟无枝酸菌基因组为模板,PCR扩增aroF基因,将aroF片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-aroF;
pSET152-aroF用XbaI和SpeI酶切,胶回收permE-aroF片段,将permE-aroF片段通过T4连接酶连接到pSET152-tyrA的SpeI位点,构建得到质粒pSET152-tyrA-aroF;pSET152-tyrA-aroF用XbaI和SpeI酶切,胶回收tyrA-aroF片段,将tyrA-aroF片段通过T4连接酶连接到pKG1132-tyrR1-2500的SpeI位点,构建得到质粒pKG1132-tyrR1-tyrA-aroF;
利用接合转移实验方法,将得到的pKG1132-tyrR1-tyrA-aroF质粒转化到步骤(1)得到的tyrR1基因缺失的重组拟无枝酸菌中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以步骤(1)的tyrR1基因缺失的重组拟无枝酸菌基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因缺失并整合tyrA-aroF基因的重组拟无枝酸菌;
(3)构建pyk基因缺失的重组拟无枝酸菌
以VA-F11/VA-R11和VA-F12/VA-R12为引物,拟无枝酸菌基因组为模板,PCR扩增pyk基因各约2.5kb的上下游同源臂,同时将上述两个pyk-up-2500和pyk-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-pyk-2500;
利用接合转移实验方法,将得到的pKG1132-pyk-2500质粒转化到步骤(2)得到的重组拟无枝酸菌中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以步骤(2)得到的重组拟无枝酸菌基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因和pyk基因缺失并整合tyrA-aroF基因的重组拟无枝酸菌;
(4)构建整合ppsA基因在pyk位点的重组拟无枝酸菌
以VA-F14/VA-R14为引物,拟无枝酸菌基因组为模板,PCR扩增ppsA基因,将ppsA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-ppsA;pSET152-ppsA用XbaI和SpeI酶切,胶回收ppsA片段,将ppsA片段通过T4连接酶连接到pKG1132-pyk-2500的SpeI位点,构建得到质粒pKG1132-pyk-ppsA;
利用接合转移实验方法,将得到的pKG1132-pyk-ppsA质粒转化到拟无枝酸菌HM-151中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-151基因组为对照,对双交换菌株进行PCR验证,验证正确的即为高产香兰素的重组拟无枝酸菌,该菌株命名为HM-153。
在本发明中,步骤(1)的拟无枝酸菌出发菌株是拟无枝酸菌HM-145。该重组菌是根据中国发明专利CN 114703113B的记载构建得到的。拟无枝酸菌HM-145表达酪氨酸解氨酶基因tal、对香豆酸-3-羟化酶基因sam5和咖啡酸甲基转移酶基因com,并缺失预苯酸脱水酶基因pheA,实现了以葡萄糖为底物发酵合成香兰素,在含有50ml发酵培养基M1的250ml锥形瓶中,实现将25g/L的葡萄糖转化为1.25g/L香兰素。
本发明还提供上述重组拟无枝酸菌或根据上述构建方法得到的重组拟无枝酸菌在产香兰素中的应用。
在本发明中,将所述重组拟无枝酸菌接种于50mL种子培养基M1中,在30C、200rpm条件下培养72h,将种子液按照质量比5%的接种量接种到含有50mL发酵培养基M1的250mL锥形瓶中,在37C、200rpm条件下发酵培养72h。其中,M1培养基配方为:葡萄糖25g/L,酵母浸粉10g/L,氯化钠0.8g/L,磷酸二氢钾5g/L,七水硫酸镁0.2g/L,氯化钙0.05g/L,其余为水,调节pH为7.2。
本发明通过在拟无枝酸菌中敲除调控蛋白基因tyrR1和丙酮酸激酶基因pyk,过表达拟无枝酸菌内源的分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroF、磷酸烯醇式丙酮酸合酶基因ppsA,使得香兰素含量达到4.72g/L,和出发菌株HM-145相比,香兰素产量提高了277.6%。其底物价格低廉,且香兰素产量较高,为工业生产进一步提高香兰素发酵产量提供技术支撑。
本发明通过基因缺失筛选,确认在调控蛋白基因tyrR1、tyrR2和tyrR3中只有tyrR1基因缺失时,具有提高拟无枝酸菌的香兰素产量的效果,与出发菌株HM-145相比,其香兰素的产量提高64.8%。进一步通过基因表达筛选,确定3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroF的序列,相比于拟无枝酸菌HM-146,整合tyrA-aroF基因的拟无枝酸菌HM-150香兰素产量提高69.42%。
附图说明
图1为拟无枝酸菌全合成香兰素通路图
图2为pSET152-permE质粒的图谱
图3为pKG1132-tyrR1-tyrA-aroF质粒的图谱
图4为pKG1132-pyk-ppsA质粒的图谱
图5为HM-150菌株PCR验证图谱,M,1Kb DNAMarker;1,3为HM150菌株用TR1-F1/tyrA-R1引物验证结果(3325bp);2,4为HM150菌株用aroF-F1/TR1-R2引物验证结果(3174bp);5为HM146菌株用TR1-F1/tyrA-R1引物验证结果(无条带);6为HM146菌株用aroF-F1/TR1-R2引物验证结果(无条带)。
图6为HM-154菌株PCR验证图谱,M,1Kb DNAMarker;1,2,3为HM154菌株用pyk-F1/ppsA-R1引物验证结果(3253bp);4为HM151菌株用pyk-F1/ppsA-R1引物验证结果(无条带);5为HM151菌株用ppsA-F1/pyk-R2引物验证结果(无条带);6,7,8为HM154菌株用ppsA-F1/pyk-R2引物验证结果(3302bp)。
具体实施方式
通过下述实施例将能够更好地理解本发明。
在本发明中,如无特殊说明,用于解释浓度的“%”均为质量百分比,用于解释比例的“:”均为质量比。
实施例中使用到的菌株和质粒见表1,合成的引物序列见表2。
表1研究中所用的菌种和质粒
表2研究中所用的引物
本发明涉及以下培养基:
LB培养基配方为:蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L。
GYM培养基配方为:葡萄糖4g/L、酵母提取物4g/L、麦芽提取物10g/L。
GYM固体培养基配方为:葡萄糖4g/L、酵母提取物4g/L、麦芽提取物10g/L、碳酸钙2g/L、琼脂粉20g/L。
M1培养基配方为:葡萄糖25g/L,酵母浸粉10g/L,氯化钠0.8g/L,磷酸二氢钾5g/L,七水硫酸镁0.2g/L,氯化钙0.05g/L,其余为水,调节pH为7.2。
以下实施例中使用的接合转移实验包括以下步骤:
(1)将拟无枝酸菌Amycolatopsis sp.(或其他工程菌株)在GYM固体培养基上进行活化,在30℃培养3-4d至长出菌落,然后接种菌株到50mL GYM液体培养基中在30℃、200rpm培养2d。
(2)将构建好的质粒热激转入E.coli ET12567(pUZ8002)菌株,涂布于含有25μg/mL氯霉素、25μg/mL卡那霉素和50μg/mL阿伯拉霉素抗性的LB固体平板,在37℃培养12h至长出单菌落,接种单菌落于含有抗性的4mL LB液体培养基中37℃、200rpm过夜培养,然后按照1%的接种量接种于20mL LB中在37℃、200rpm培养4-5h至OD600为0.4-0.6。
(3)分别取拟无枝酸菌Amycolatopsis sp.菌液2mL及携带目标质粒的E.coliET12567(pUZ8002)菌液1mL,5000g离心1min,用无抗LB清洗两遍,加入100μL无抗LB培养基,将拟无枝酸菌和E.coli ET12567(pUZ8002)按体积比7:1混合,将混合好的菌液取30μL点在无抗GYM固体培养基上30℃,14h正置培养。
(4)将长出的菌斑刮下,涂布于含有50μg/mL阿伯拉霉素和25μg/mL萘啶酮酸溶液GYM固体培养基中,在30℃培养4d至长出单菌落,然后对单菌落进行PCR验证。
实施例1:构建tyrR基因缺失的拟无枝酸菌
在NCBI网站上根据拟无枝酸菌Amycolatopsis sp.ATCC 39116菌株的基因序列比对出拟无枝酸菌中调控蛋白tyrR1、tyrR2、tyrR3的序列,其中tyrR1的基因序列如SEQ IDNO:1所示,其编码的蛋白质登录号为WP_020416143.1,tyrR2的基因序列如SEQ ID NO:6所示,其编码的蛋白质登录号为WP_020422712.1,tyrR3的基因序列如SEQ ID NO:7所示,其编码的蛋白质登录号为WP_020419034.1。
1、构建tyrR基因敲除的质粒pKG1132-tyrR1-2500、pKG1132-tyrR2-2500、pKG1132-tyrR3-2500
以VA-F2/VA-R2和VA-F3/VA-R3为引物,拟无枝酸菌基因组为模板,PCR扩增tyrR1基因各约2.5kb的上下游同源臂。同时将得到的两个tyrR1-up-2500和tyrR1-down-2500片段通过Assembly试剂盒连接到pKG1132质粒(Nadja Barton,et al.,Enabling thevalorization of guaiacol-based lignin:Integrated chemical and biochemicalproduction of cis,cis-muconic acid using metabolically engineeredAmycolatopsis sp ATCC 39116,Metabolic Engineerings45(2018)200-210)的HindIII/EcoRI位点,构建得到质粒pKG1132-tyrR1-2500。
以VA-F4/VA-R4和VA-F5/VA-R5为引物,拟无枝酸菌基因组为模板,PCR扩增tyrR2基因各约2.5kb的上下游同源臂。同时将上述两个tyrR2-up-2500和tyrR2-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-tyrR2-2500。
以VA-F6/VA-R6和VA-F7/VA-R7为引物,拟无枝酸菌基因组为模板,PCR扩增tyrR3基因各约2.5kb的上下游同源臂。同时将上述两个tyrR3-up-2500和tyrR3-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-tyrR3-2500。
2、构建tyrR基因缺失的拟无枝酸菌
利用接合转移实验方法,分别将得到的pKG1132-tyrR1-2500、pKG1132-tyrR2-2500、pKG1132-tyrR3-2500质粒转化到拟无枝酸菌HM-145中,在有阿伯拉霉素抗性(终浓度为50μg/mL)的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株。将所得单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-145基因组为对照,对双交换菌株进行PCR验证,tyrR1基因缺失验证引物为TR1-F1/TR1-R1和TR1-F2/TR1-R2,tyrR2基因缺失验证引物为TR2-F1/TR2-R1和TR2-F2/TR2-R2,tyrR3基因缺失验证引物为TR3-F1/TR3-R1和TR3-F2/TR3-R2。PCR验证正确的条带送测序,测序正确的即为敲除tyrR基因的基因缺失拟无枝酸菌,分别将敲除tyrR1、tyrR2、tyrR3基因的菌株命名为HM-146、HM-147、HM-148。
3、出发菌株拟无枝酸菌HM-145与HM-146、HM-147、HM-148菌株的发酵对比
分别用拟无枝酸菌HM-145与HM-146、HM-147、HM-148菌株进行发酵实验,实验方法如下:
分别接种上述菌株于50mL种子培养基M1中,在30℃、200rpm培养72h,将种子液按照5%的接种量接种到含有50mL发酵培养基M1的250mL锥形瓶中,在37℃、200rpm条件下发酵培养72h。发酵停止后,测定发酵液中香兰素的浓度。
香兰素含量测定方法:参照中国发明专利申请CN 113789292A中记载的方法,结果如表3所示。
表3拟无枝酸菌HM-145及其工程菌株的摇瓶发酵实验结果
以上结果可以看出,tyrR1基因缺失型拟无枝酸菌HM-146提高了葡萄糖合成香兰素的产量,从出发菌株HM-145的1.25g/L提升到2.06g/L,提升了64.8%。tyrR2基因缺失型拟无枝酸菌HM-147的香兰素产量相比于HM-145菌株,仅仅提高了4.8%。tyrR3基因缺失型拟无枝酸菌HM-147的香兰素产量相比于HM-145菌株,降低13.6%。由此可见,尽管tyrR1、tyrR2和tyrR3都是拟无枝酸菌的调控蛋白基因,但是只有敲除tyrR1基因才能起到以葡萄糖为底物的香兰素产量,而tyrR2和tyrR3均不具备类似技术效果。
因此,在tyrR1基因缺失型拟无枝酸菌HM-146的基础上进行后续的操作。
实施例2:构建整合tyrA、aroF基因的拟无枝酸菌HM-150
在NCBI网站上根据拟无枝酸菌Amycolatopsis sp.ATCC 39116菌株的基因序列比对出拟无枝酸菌中分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroG、aroF的序列,其中tyrA的基因序列如SEQ ID NO:2所示,其编码的蛋白质登录号为WP_020419478.1,aroF的基因序列如SEQ ID NO:3所示,其编码的蛋白质登录号为WP_020418838.1,aroG的基因序列如SEQ ID NO:8所示,其编码的蛋白质登录号为WP_026153352.1。
1、构建整合质粒pSET152-tyrA-aroG、pSET152-tyrA-aroF、pKG1132-tyrR1-tyrA-aroG、pKG1132-tyrR1-tyrA-aroF
以VA-F1/VA-R1为引物,pKC1139-permE(根据中国发明专利CN 114703113B的记载构建得到)为模板,PCR扩增permE片段,将permE片段通过Assembly试剂盒连接到pSET152质粒的PvuI/EcoRV位点,构建得到质粒pSET152-permE。pSET152-permE质粒图谱如图1所示。
以VA-F8/VA-R8为引物,拟无枝酸菌基因组为模板,PCR扩增tyrA基因,将tyrA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-tyrA。
以VA-F9/VA-R9为引物,拟无枝酸菌基因组为模板,PCR扩增aroG基因,将aroG片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-aroG。
以VA-F10/VA-R10为引物,拟无枝酸菌基因组为模板,PCR扩增aroF基因,将aroF片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-aroF。
pSET152-aroG用XbaI和SpeI酶切,胶回收permE-aroG片段,将permE-aroG片段通过T4连接酶连接到pSET152-tyrA的SpeI位点,构建得到质粒pSET152-tyrA-aroG。
pSET152-aroF用XbaI和SpeI酶切,胶回收permE-aroF片段,将permE-aroF片段通过T4连接酶连接到pSET152-tyrA的SpeI位点,构建得到质粒pSET152-tyrA-aroF。
pSET152-tyrA-aroG用XbaI和SpeI酶切,胶回收tyrA-aroG片段,将tyrA-aroG片段通过T4连接酶连接到pKG1132-tyrR1-2500的SpeI位点,构建得到质粒pKG1132-tyrR1-tyrA-aroG。
pSET152-tyrA-aroF用XbaI和SpeI酶切,胶回收tyrA-aroF片段,将tyrA-aroF片段通过T4连接酶连接到pKG1132-tyrR1-2500的SpeI位点,构建得到质粒pKG1132-tyrR1-tyrA-aroF,pKG1132-tyrR1-tyrA-aroF质粒图谱如图2所示。
2、构建整合tyrA-aroG/tyrA-aroF基因的拟无枝酸菌HM-149、HM-150
利用接合转移实验方法,分别将得到的pKG1132-tyrR1-tyrA-aroG、pKG1132-tyrR1-tyrA-aroF质粒转化到拟无枝酸菌HM-146中,在有阿伯拉霉素抗性(终浓度为50μg/mL)的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株。将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-146基因组为对照,对双交换菌株进行PCR验证,整合tyrA-aroG基因验证引物为TR1-F1/tyrA-R1和aroG-F1/TR1-R2。整合tyrA-aroF基因验证引物为TR1-F1/tyrA-R1和aroF-F1/TR1-R2。PCR验证正确的条带送测序,测序正确的即为整合tyrA-aroG/tyrA-aroF基因的拟无枝酸菌HM-149、HM-150,HM-150菌株的PCR验证结果如图5所示。
3、HM-146与HM-149、HM-150菌株的发酵对比
分别用拟无枝酸菌HM-146与HM-149、HM-150菌株进行发酵实验,发酵实验方法同实施例1。发酵结果如下表4所示。
表4拟无枝酸菌HM-146与HM-149、HM-150菌株的摇瓶发酵实验结果
以上结果可以看出,相比于拟无枝酸菌HM-146,整合tyrA-aroG基因的拟无枝酸菌HM-149香兰素产量提高22.81%,整合tyrA-aroF基因的拟无枝酸菌HM-150香兰素产量提高69.42%。由此可见,尽管aroG和aroF都是3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因,但aroF基因的表达对于提高拟无枝酸菌以葡萄糖为底物发酵产香兰素产量具有显著效果,而aroG尽管也能一定程度提高香兰素产量,但产量提高幅度不及预期。
因此,在整合tyrA-aroF基因的拟无枝酸菌HM-150的基础上进行后续的操作。
实施例3:构建pyk基因缺失的拟无枝酸菌
为了进一步提高拟无枝酸菌以葡萄糖为底物发酵产香兰素产量,在NCBI网站上根据拟无枝酸菌Amycolatopsis sp.ATCC 39116菌株的基因序列比对出拟无枝酸菌中丙酮酸激酶基因pyk的序列,pyk的基因序列如SEQ ID NO:4所示,其编码的蛋白质登录号为WP_027935993.1。
1、构建pyk基因敲除的质粒pKG1132-pyk-2500
以VA-F11/VA-R11和VA-F12/VA-R12为引物,拟无枝酸菌基因组为模板,PCR扩增pyk基因各约2.5kb的上下游同源臂。同时将上述两个pyk-up-2500和pyk-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-pyk-2500。
2、构建pyk基因缺失的拟无枝酸菌HM-151
利用接合转移实验方法,将得到的pKG1132-pyk-2500质粒转化到拟无枝酸菌HM-150中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株。将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-150基因组为对照,对双交换菌株进行PCR验证,pyk基因缺失验证引物为pyk-F1/pyk-R1和pyk-F2/pyk-R2。PCR验证正确的条带送测序,测序正确的即为敲除pyk基因的基因缺失拟无枝酸菌HM-151。
3、HM-150与HM-151菌株的发酵对比
分别用拟无枝酸菌HM-150与HM-151菌株进行发酵实验,发酵实验方法同实施例1。发酵结果如下表3所示。
表3拟无枝酸菌HM-150与HM-151菌株的摇瓶发酵实验结果
以上结果可以看出,pyk基因缺失型拟无枝酸菌HM-151提高了香兰素的产量,和拟无枝酸菌HM-150相比,提高10.32%。因此,在pyk基因缺失型拟无枝酸菌HM-151的基础上进行后续的操作。
实施例4:构建整合ppsA基因的拟无枝酸菌HM-154
为了进一步提高拟无枝酸菌以葡萄糖为底物发酵产香兰素产量,在NCBI网站上根据拟无枝酸菌Amycolatopsis sp.ATCC 39116菌株的基因序列比对出拟无枝酸菌中转酮酶基因tktA、磷酸烯醇式丙酮酸合酶基因ppsA的序列,其中ppsA的基因序列如SEQ ID NO:5所示,其编码的蛋白质登录号为WP_020420172.1。
1、构建整合质粒pSET152-tktA、pSET152-ppsA、pKG1132-pyk-ppsA
以VA-F13/VA-R13为引物,拟无枝酸菌基因组为模板,PCR扩增tktA基因,将tktA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-tktA。以VA-F14/VA-R14为引物,拟无枝酸菌基因组为模板,PCR扩增ppsA基因,将ppsA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-ppsA。
pSET152-ppsA用XbaI和SpeI酶切,胶回收ppsA片段,将ppsA片段通过T4连接酶连接到pKG1132-pyk-2500的SpeI位点,构建得到质粒pKG1132-pyk-ppsA。
2、构建整合ppsA基因的拟无枝酸菌HM-154
利用接合转移实验方法,分别将得到的pSET152-tktA、pSET152-ppsA质粒转化到拟无枝酸菌HM-151中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落分别为整合tktA基因的菌株HM-152,整合ppsA基因的HM-153。
利用接合转移实验方法,将得到的pKG1132-pyk-ppsA质粒转化到拟无枝酸菌HM-151中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株。将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-151基因组为对照,对双交换菌株进行PCR验证,验证引物为pyk-F1/ppsA-R1和ppsA-F1/pyk-R2,PCR验证正确的条带送测序,测序正确的即为整合ppsA基因的拟无枝酸菌HM-154,HM-154菌株的PCR验证结果如图6所示。
3、HM-151与HM-152、HM-153、HM-154菌株的发酵对比
分别用拟无枝酸菌HM-151与HM-152、HM-153菌株进行发酵实验,发酵实验方法同实施例1。发酵结果如下表4所示。
表4拟无枝酸菌HM-151与HM-152、HM-153菌株的摇瓶发酵实验结果
以上结果可以看出,整合tktA基因的菌株HM-152的香兰素产量相比于HM-151菌株,降低5.2%,整合ppsA基因的菌株HM-153香兰素产量相比于HM-151菌株,提高21.82%。
过表达tktA能够增加前体赤藓糖-4-磷酸(E4P)的通量,而过表达ppsA增加前体磷酸烯醇式丙酮酸(PEP)的通量,E4P和PEP是合成香兰素的重要前体物质,如图1所示。
结果表明,尽管E4P和PEP是合成香兰素的重要前体物质,但拟无枝酸菌中转酮酶基因tktA的表达不能提高拟无枝酸菌以葡萄糖为底物发酵产香兰素产量,而磷酸烯醇式丙酮酸合酶基因ppsA则能够进一步提高拟无枝酸菌以葡萄糖为底物发酵产香兰素产量,推测在拟无枝酸菌中tktA不是提高E4P通量的关键基因。
综上所述,本发明通过在拟无枝酸菌工程菌株中敲除调控蛋白基因tyrR1和丙酮酸激酶基因pyk,并过表达拟无枝酸菌内源的分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroF和磷酸烯醇式丙酮酸合酶基因ppsA,获得HM-154菌株,经过发酵实验,HM-154菌株的香兰素产量为4.72g/L,和HM-151菌株相比,香兰素产量提高了22.6%,和出发菌株HM-145相比,香兰素产量提高了277.6%。
Claims (6)
1. 拟无枝酸菌(Amycolatopsis sp.)调控蛋白tyrR1基因缺失在提高拟无枝酸菌(Amycolatopsis sp.)以葡萄糖为底物发酵产香兰素的香兰素产量中的应用,所述tyrR1的基因序列如SEQ ID NO:1所示,其编码的蛋白质登录号为WP_020416143.1。
2. 过表达tyrA-aroF基因并缺失调控蛋白tyrR1基因在提高拟无枝酸菌以葡萄糖为底物发酵产香兰素的香兰素产量中的应用,所述tyrA是分支酸变位酶/预苯酸脱氢酶双功能酶基因,其核酸序列如SEQ ID NO:2所示,其编码的蛋白质登录号为WP_020419478.1,所述aroF基因是3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因,aroF的基因序列如SEQID NO:3所示,其编码的蛋白质登录号为WP_020418838.1,所述tyrR1的基因序列如SEQ IDNO:1所示,其编码的蛋白质登录号为WP_020416143.1。
3. 一种高产香兰素产量的重组拟无枝酸菌,所述重组拟无枝酸菌敲除了拟无枝酸菌(Amycolatopsis sp.)HM-145的调控蛋白基因tyrR1和丙酮酸激酶基因pyk,过表达拟无枝酸菌内源的分支酸变位酶/预苯酸脱氢酶双功能酶基因tyrA、3-脱氧-D-***庚酮糖酸-7-磷酸(DAHP)合成酶基因aroF和磷酸烯醇式丙酮酸合酶基因ppsA,基因tyrR1的核苷酸序列如SEQ ID NO:1所示,基因tyrA的核苷酸序列如SEQ ID NO:2所示,基因aroF的核苷酸序列如SEQ ID NO:3所示,基因pyk的核苷酸序列如SEQ ID NO:4所示,基因ppsA的核苷酸序列如SEQ ID NO:5所示。
4.权利要求3所述的重组拟无枝酸菌的构建方法,所述方法包括以下步骤:
(1)构建tyrR1基因缺失的重组拟无枝酸菌
以VA-F2/VA-R2和VA-F3/VA-R3为引物,拟无枝酸菌HM-145基因组为模板,PCR扩增tyrR1基因的上下游同源臂,同时将所得的两个tyrR1-up-2500和tyrR1-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-tyrR1-2500;
利用接合转移实验方法,将得到的pKG1132-tyrR1-2500质粒转化到拟无枝酸菌出发菌株中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-145基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因缺失的重组拟无枝酸菌;
(2)构建整合tyrA、aroF基因的重组拟无枝酸菌
以VA-F1/VA-R1为引物,pKC1139-permE为模板,PCR扩增permE片段,将permE片段通过Assembly试剂盒连接到pSET152质粒的PvuI/EcoRV位点,构建得到质粒pSET152-permE;以VA-F8/VA-R8为引物,拟无枝酸菌基因组为模板,PCR扩增tyrA基因,将tyrA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-tyrA;以VA-F10/VA-R10为引物,拟无枝酸菌基因组为模板,PCR扩增aroF基因,将aroF片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-aroF;
pSET152-aroF用XbaI和SpeI酶切,胶回收permE-aroF片段,将permE-aroF片段通过T4连接酶连接到pSET152-tyrA的SpeI位点,构建得到质粒pSET152-tyrA-aroF;pSET152-tyrA-aroF用XbaI和SpeI酶切,胶回收tyrA-aroF片段,将tyrA-aroF片段通过T4连接酶连接到pKG1132-tyrR1-2500的SpeI位点,构建得到质粒pKG1132-tyrR1-tyrA-aroF;
利用接合转移实验方法,将得到的pKG1132-tyrR1-tyrA-aroF质粒转化到步骤(1)得到的tyrR1基因缺失的重组拟无枝酸菌中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以步骤(1)的tyrR1基因缺失的重组拟无枝酸菌基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因缺失并整合tyrA-aroF基因的重组拟无枝酸菌;
(3)构建pyk基因缺失的重组拟无枝酸菌
以VA-F11/VA-R11和VA-F12/VA-R12为引物,拟无枝酸菌基因组为模板,PCR扩增pyk基因上下游同源臂,同时将上述两个pyk-up-2500和pyk-down-2500片段通过Assembly试剂盒连接到pKG1132质粒的HindIII/EcoRI位点,构建得到质粒pKG1132-pyk-2500;
利用接合转移实验方法,将得到的pKG1132-pyk-2500质粒转化到步骤(2)得到的重组拟无枝酸菌中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以步骤(2)得到的重组拟无枝酸菌基因组为对照,对双交换菌株进行PCR验证,验证正确的即为tyrR1基因和pyk基因缺失并整合tyrA-aroF基因的重组拟无枝酸菌;
(4)构建整合ppsA基因在pyk位点的重组拟无枝酸菌
以VA-F14/VA-R14为引物,拟无枝酸菌基因组为模板,PCR扩增ppsA基因,将ppsA片段通过Assembly试剂盒连接到pSET152-permE质粒的BamHI/NsiI位点,构建得到质粒pSET152-ppsA;pSET152-ppsA用XbaI和SpeI酶切,胶回收ppsA片段,将ppsA片段通过T4连接酶连接到pKG1132-pyk-2500的SpeI位点,构建得到质粒pKG1132-pyk-ppsA;
利用接合转移实验方法,将得到的pKG1132-pyk-ppsA质粒转化到拟无枝酸菌HM-151中,在有阿伯拉霉素抗性的GYM固体培养基上长出的单菌落即为发生同源单交换的菌株;将单交换菌株在无抗GYM培养基中进行传代培养,在无抗GYM平板上生长且阿伯拉霉素抗性平板不长的菌株即为发生同源双交换的菌株,以拟无枝酸菌HM-151基因组为对照,对双交换菌株进行PCR验证,验证正确的即为高产香兰素的重组拟无枝酸菌;
VA-F2:GGCCAGTGCCAAGCTTGGGTTGCGGTCGACCACGATCA
VA-R2:CCTTGATCACTAGTGGACCGCCCGCTGATCCACCAT
VA-F3:CACTAGTGATCAAGGTGCCGCGCCGGTAG
VA-R3:ACATGATTACGAATTCTGGGATAGCACCGGAGGAGGGT
VA-F1:CTGTTGGGAAGGGCGTCTAGAAGCCCGACCCGAGCAC
VA-R1:GATTACGAATTCGATACTAGTACCGATACAATTAAAGGCTCCTTTTG
VA-F8:CCGGTTGGTAGGATCCAAGGAGGCAACAAGGTGATCGGGCTCGGGTTGATCG
VA-R8:TTTTTGGAGATTTTATGCATTTACGGCACCTCGCCGATGGCG
VA-F10:CCGGTTGGTAGGATCCAAGGAGGCAACAAGATGTCTCCCAGCGCCTCGACAC:
VA-R10:TTTTTGGAGATTTTATGCATCTACCGCCGAGCAGCCACAGCC
VA-F11:CGGCCAGTGCCAAGCTGGGCAAGTACGAGCAGGGCATC
VA-R11:CTCACCGAACTAGTATCTTCGCGCGTCGGCTCACGT
VA-F12:TACTAGTTCGGTGAGGACGACCACGCCTGA
VA-R12:ACATGATTACGAATTCCACCGGGGCAAACGGCTCCACA。
5.权利要求3所述的重组拟无枝酸菌或根据权利要求4所述构建方法得到的重组拟无枝酸菌在产香兰素中的应用。
6.根据权利要求5所述的应用,其特征在于将所述重组拟无枝酸菌接种于50mL种子培养基M1中,在30℃、200rpm条件下培养72h,将种子液按照质量比5%的接种量接种到含有50mL发酵培养基M1的250mL锥形瓶中,在37℃、200rpm条件下发酵培养72h;所述M1培养基配方为:葡萄糖25g/L,酵母浸粉10g/L,氯化钠0.8g/L,磷酸二氢钾5g/L,七水硫酸镁0.2g/L,氯化钙0.05g/L,其余为水,调节pH为7.2。
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