CN114561309B - 一种亚细胞区室化生产衣康酸的解脂耶氏酵母工程菌及应用 - Google Patents
一种亚细胞区室化生产衣康酸的解脂耶氏酵母工程菌及应用 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域,具体涉及一种区室化生产衣康酸的解脂耶氏酵母工程菌构建及应用。所述基因工程菌以解脂耶氏酵母为出发菌株,将顺‑乌头酸脱羧酶编码基因通过增强型过氧化物酶体定位信号ePTS1在过氧化物酶体中进行定位表达获得。通过引入ePTS1将衣康酸在过氧化物酶体中进行区室化表达后,衣康酸产量有明显提升,提高了47倍左右;采用厨房废油(YPO)为培养基,衣康酸产量提高了108倍左右。可见,定位改造后的基因工程菌株,更适合工业应用,可降低生产成本,提高生产效率;而采用厨房废油作为培养基不仅实现废物利用,降低成本,而且产量远远高于常用普通培养基。
Description
技术领域:
本发明属于分子生物学技术领域,具体涉及一种区室化生产衣康酸的解脂耶氏酵母工程菌构建及应用。
背景技术:
解脂耶氏酵母(Yarrowia lipolytica)是一种非常规的产油酵母,由于其充足的前体供应,适于生产各种源于乙酰辅酶A的下游化合物。但以葡萄糖为碳源时,产生乙酰辅酶A的同时会伴随二氧化碳(CO2)的释放,造成碳损失及得率的降低。而以脂肪酸作为底物时,脂肪酸转化成酰基辅酶A后进入过氧化物酶体,经过β-氧化产生乙酰辅酶A,这一过程不产生CO2,不存在碳源的损失,具有更高的碳转化率。近年来越来越多的研究表明,特定酶或者代谢途径的亚细胞定位不仅有助于提高产物转化效率,而且能够起到消除竞争性代谢抑制的作用。
区室化工程是利用细胞中细胞器独特的生理生化功能进行代谢工程与合成生物学研究等。同时,由于细胞器膜的阻隔,代谢途径的细胞器或者亚细胞定位能够避免胞质环境的代谢竞争,提高产物合成效率。细胞器有许多独特的生理和代谢环境,比如内质网与脂质的形成有关,过氧化物酶体参与脂肪酸的β-氧化且具有较高的蛋白质浓度,细胞核富含DNA等等。
衣康酸(C5H6O4)学名为甲叉琥珀酸,亚甲基丁二酸,是一种不饱和二元有机酸。它含有不饱和双键,具有活泼的化学性质,可进行自身间的聚合,也能与其他单体如丙烯腈等聚合,微溶于苯、氯仿、***、石油醚、二硫化碳,溶于水、乙醇、丙酮;能进行各种加成反应,酯化反应和聚合反应,是化学合成工业的重要原料,也是化工生产的重要原料。其在酸性、中性和中等碱性条件下均表现为性质稳定,可用于树脂、纤维、洗涤剂、清洁剂和生物活性成分等工业产品的合成。衣康酸的工业生产有化学合成和生物合成两种方式,化学合成主要是利用化工方法从石油化工产品中制备,然而这种方法存在原料利用率低、产品不易分离纯化的问题,使其在大规模工业化生产中受到了限制。衣康酸的生物合成方法是指通过对微生物代谢途径的遗传修饰来完成目标产物的制备。其中,土曲霉、玉米黑粉菌、念珠菌属、毕赤酵母等真菌天然生产衣康酸的能力较强。然而,真菌具有菌丝表型,这是因为它们对剪切应力的耐受性较差。高度分支的菌丝丝在发酵过程中产生高的肉汤粘度,这给常规搅拌槽发酵罐中的混合和通气过程带来了重大挑战。此外,它们发酵需要添加碱以维持中性pH条件,这也增加了培养期间细菌污染的可能性。解脂耶氏酵母本身并不能产生衣康酸,但它对衣康酸具有较高的耐受性。因此近年来,利用代谢工程及合成生物学改造解脂耶氏酵母异源生产衣康酸成为了相关研究的热点。
发明内容:
本发明所要解决的技术问题就是解决在解脂耶氏酵母中异源合成衣康酸产量低的问题,提供一株导入顺-乌头酸脱羧酶(CAD)基因的解脂耶氏酵母基因工程菌以及该菌株在异源合成衣康酸中的用途,其能够合成衣康酸并通过代谢工程或合成生物学手段提高衣康酸产量。
本发明提供的技术方案之一:是一种解脂耶氏酵母基因工程菌,所述基因工程菌以解脂耶氏酵母为出发菌株,通过导入顺-乌头酸脱羧酶编码基因来获得。
所述顺-乌头酸脱羧酶编码基因,其核苷酸如序列表SEQ ID NO.1所示;是通过土曲霉HAT418的基因组上两段不含内含子的基因片段进行融合获得的。
进一步地,所述CAD基因是通过质粒在宿主细胞中表达;
更进一步地,所述质粒为pYLEX1;
进一步地,所述CAD基因通过增强型过氧化物酶体定位信号(ePTS1)在过氧化物酶体中进行定位表达;
更进一步地,将ePTS1编码序列连接到CAD基因序列的后面,构建重组质粒pYLEX1-CAD-ePTS1,并将重组质粒导入解脂耶氏酵母出发菌株中进行表达;
更进一步地,所述ePTS1编码序列如序列表SEQ ID NO.2所示(ctgggccgaggacgacgatccaagctg);
优选地,所述解脂耶氏酵母出发菌株为解脂耶氏酵母(Yarrowia lipolytica)Po1g ku70Δ。
本发明提供的技术方案之二,是上述解脂耶氏酵母基因工程菌的应用,特别是在生产衣康酸中的应用;
进一步地,采用上述解脂耶氏酵母基因工程菌发酵生产衣康酸的方法如下:
将工程菌种子液按1%接种量接种至YPD培养基中,28-30℃,200-220r/min条件培养15-18h,再转接至YPO培养基中使OD600达到0.1左右,28-30℃,200-220r/min条件下发酵6天。
所述YPD培养基组成成分为:蛋白胨20g/L,酵母浸粉10g/L,葡萄糖20g/L,余量为水,115-121℃,灭菌20min。
所述YPO培养基组成成分为:蛋白胨18-22g/L,酵母浸粉8-12g/L,厨房废油1.1-1.2%,吐温80 0.18-0.22%,余量为水,115-121℃,灭菌20min。
有益效果:
本发明通过在解脂耶氏酵母中异源引入CAD基因证实了生产衣康酸的可能(宿主解脂耶氏酵母Po1g ku70Δ本身不能生产衣康酸),产量为33.12mg/L。本发明还利用绿色荧光蛋白和尼罗红亲脂性的特性验证了增强型过氧化物酶体定位信号ePTS1在解脂耶氏酵母中是起到作用的。同时本发明第一次将CAD基因在解脂耶氏酵母的过氧化物酶体中进行异源定位表达,通过消耗厨房废油的脂肪酸来生产衣康酸。
结果表明,通过引入ePTS1将衣康酸在过氧化物酶体中进行区室化表达后,与单纯异源表达CAD基因的菌株相比衣康酸产量有明显提升,产量为1.58g/L,提高了47倍左右;采用厨房废油(YPO)为培养基,相对于普通YPD培养基,衣康酸产量提高了108倍左右。可见,定位改造后的基因工程菌株,更适合工业应用,可降低生产成本,提高生产效率;而采用厨房废油作为培养基不仅实现废物利用,降低成本,而且产量远远高于常用普通培养基。
附图说明:
图1衣康酸生产途径示意图;
图2为CAD基因重组质粒的构建图;
图3为验证基因片段凝胶电泳图
其中,图a为重组质粒pYLEX1-CAD验证图;图b为重组菌株Po1gku70Δ-CAD-ePTS1验证图;
图4为显微镜下的荧光图
其中,a图为酵母在488nm下显现的绿色荧光,b图为酵母在561nm下显现的红色荧光,c图为a、b重叠结合图;
图5为不同菌株及培养基生产衣康酸的对比产量图。
具体实施方式:
下面结合实施例对本发明的技术内容做进一步说明。需要说明的是,在不冲突的情况下,本发明中的实施例仅为本发明中的一部分实施例,而不是全部的实施例。基于本发明的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的其他实施例,都属于本发明保护范围之内。
本发明所使用的解脂耶氏酵母Po1gKU70△为现有技术,是在解脂耶氏酵母Po1g菌株中敲除KU70基因获得的,其构建方法参考文献Genetic engineering of anunconventional yeast for renewable biofuel and biochemical production.Journalof Visualized Experiments,2016,115,e54371。所述解脂耶氏酵母Po1g菌株购买自中国台湾的Yeastern Biotech Co.公司。
本发明所使用的pYLEX1质粒,购自Yeastern Biotech Co.,Ltd.。所述质粒带有营养缺陷筛选基因亮氨酸表达盒、标记基因Amp、强启动子hp4d以及终止子XPR2 term。
本发明测定发酵液中衣康酸的方法为:
对发酵完的培养液进行离心,取2mL发酵上清液加入1.5mL 10%
HCl-CH3OH溶液,62℃酯化3h,然后加入2mL正己烷,剧烈振荡1min,使衣康酸二甲酯溶解。离心(6000r/min,5min)后,将上层有机相倒入另一个干净的瓶中进行GC-MS检测衣康酸二甲酯含量。
取2mL 100mg/L衣康酸标准品按上述方法进行甲酯化,GC-MS检测衣康酸标准品甲酯化后所得衣康酸二甲酯含量。再取100mg/L衣康酸二甲酯标准品进行GC-MS检测衣康酸二甲酯含量。最终将两者衣康酸二甲酯含量进行换算得到按上述方法甲酯化的甲酯率,根据该甲酯化率和发酵样品甲酯化后的衣康酸二甲酯含量,即能算得样品中的衣康酸产量。
以下通过具体实施例对本发明作进一步地解释说明。
实施例1:产衣康酸的解脂耶氏酵母菌株的构建
(1)根据SEQ ID NO.1所示的CAD基因核苷酸序列进行基因合成,设计引物对获得的CAD基因片段进行扩增。
PCR反应体系为(重组酶来自南京诺唯赞生物科技有限公司):
组分 | 体系 |
片段 | 1μL |
CAD1-F/CAD2-R | 1μL/个 |
2×phanta Max Master Mix | 10μL |
H2O | ~20μL |
PCR反应条件:95℃5min;95℃30s;72℃30s;72℃1min;72℃10min,30个循环。
(2)将扩增后的CAD基因进行切胶纯化,使用限制性内切酶Pml1将原始质粒pYLEX1酶切为线性质粒,然后将CAD基因片段整合至线性质粒,获得重组质粒pYLEX1-CAD,具体构建示意图为图2。
(3)用引物PYL-F/R对重组质粒pYLEX1-CAD进行PCR验证,得到的片段大小为1870bp的条带(图3a)。
(4)将(2)中重组质粒pYLEX1-CAD用Spe1酶切线性化后,用醋酸锂转化法将线性化片段转化入Po1g ku70Δ中,在亮氨酸缺陷板YNB上筛选得到重组后的解脂耶氏酵母菌株Po1g ku70Δ-CAD。提取转化子菌株Po1g ku70Δ-CAD的基因组DNA,并以此为模板,进行PCR验证,引物与(3)中使用的一致,得到的结果也一致。此结果说明线性化重组质粒已经成功整合到出发菌株的染色体基因组上,即得基因工程菌株Po1g ku70Δ-CAD。
实施例2将CAD基因定位于解脂耶氏酵母过氧化物酶体中
(1)ePTS1序列:ctgggccgaggacgacgatccaagctg,将此序列以引物形式加到hrGFPO序列(SEQ ID NO.3)终止子前获得hrGFPO-ePTS1基因片段,再将所述片段连接至质粒pYLEX1,构建重组质粒pYLEX1-hrGFPO-ePTS1,然后转化解脂耶氏酵母菌株Po1g ku70Δ获得重组菌株。
验证正确后挑取转化子在含有50mL YPD三角瓶中于30℃摇床中培养24小时,加入1mg/mL的丙酮-尼罗红溶液于培养物中,室温下避光培养1小时。染色后的细胞用生理盐水洗涤,再用磷酸钾缓冲液(pH7.4)重悬细胞。
将重悬后的细胞液直接镀于载玻片,盖上盖玻片放在激光扫描共焦显微镜下观察分别位于488nm时的GFP和561nm时的尼罗红。在这里,我们选择使用尼罗红染色来确定解脂耶氏酵母中过氧化物酶体的定位,因为它是一种可以染色胞内脂质的干性染料。绿色荧光与红色荧光几乎可以重叠为黄色,由此可以证明ePTS1在解脂耶氏酵母中可以起到定位过氧化物酶体的作用(详见图4:a图为酵母在488nm下显现的绿色荧光,b图为酵母在561nm下显现的红色荧光,c图为a、b重叠结合图)。
(2)将ePTS1序列以引物形式加到SEQ ID NO.1所示的CAD基因终止子序列前获得CAD-ePTS1基因片段,采用实施例1步骤(2)同样的方法将CAD-ePTS1基因片段整合至pYLEX1线性质粒,构建重组质粒pYLEX1-CAD-ePTS1,经大肠杆菌扩增后转化解脂耶氏酵母Po1gku70Δ,验证正确的菌株命名为Po1g ku70Δ-CAD-ePTS1(图3b)。
分别以hrGFPO或CAD编码基因为模板,扩增hrGFPO-ePTS1或CAD-ePTS1基因片段,所用引物如下:
实施例3解脂耶氏酵母生产衣康酸
(1)将验证正确的菌株Po1g ku70Δ-CAD-ePTS1、Po1g ku70Δ-CAD分别作为生产菌株,挑取转化子于5mL YPD试管中培养24小时,再取250μL的培养液于25mL YPD三角瓶中培养16小时,取适量培养液于50mL YPO或YPD培养基中,使OD600达到0.1,发酵4天(pH不做控制,均在30℃,220r/min的振荡培养箱中进行)。
(2)对发酵完的培养液进行离心,取2mL发酵上清液加入1.5mL 10%HCl-CH3OH溶液,62℃酯化3h,然后加入2mL正己烷,剧烈振荡1min,使衣康酸二甲酯溶解。离心(6000r/min,5min)后,将上层有机相倒入另一个干净的瓶中进行检测。按上述方法取3份2mL100mg/L衣康酸标准品进行甲酯化,用100mg/L衣康酸二甲酯标准品分析的面积换算为GC-MS分析的平均面积。
(3)使用配有HP-5MS色谱柱(30m×0.25mm×0.25微米,安捷伦,美国加利福尼亚州圣克拉拉)的Agilent 7890A GC和5975C MSD,通过GC-MS分析0.6μL有机相。GC烘箱温度最初在60℃保持2分钟,然后以10℃/分钟的速率升至250℃并保持9分钟。分流比为10:1。氦气用作载气,入口压力为13.8psi。注射器温度保持在250℃,离子源温度设定为220℃。使用增强型数据分析软件(Agilent,Santa Clara,CA,USA)进行最终数据分析。结果如下表及图5:
采用不同菌株和培养基生产衣康酸第4天的产量(mg/L)
本发明通过在解脂耶氏酵母中异源引入CAD基因证实了生产衣康酸的可能,产量为33.12mg/L。同时本发明第一次将CAD基因在解脂耶氏酵母的过氧化物酶体中进行异源定位表达,通过消耗厨房废油的脂肪酸来生产衣康酸,与单纯异源表达CAD基因的菌株相比衣康酸产量有明显提升,产量为1.58g/L,提高了48倍。具体结果见图5,图中第二行数据为菌株Po1g ku70Δ-CAD-ePTS1在YPO培养基发酵所得,第三行数据/>为菌株Po1g ku70Δ-CAD-ePTS1在YPD培养基发酵所得,第四行数据/>为菌株Po1g ku70Δ-CAD在YPO培养基发酵所得。
经测定,使用厨房废油区室化定位过氧化物酶体对于在解脂耶氏酵母中生产衣康酸产量有极大提高,说明了此方法既降低了成本,又提高了产量。我们相信,通过对上述研究的进一步开展,解脂耶氏酵母在生产羧酸方面必定会扮演越来越重要的角色,最终成为工业生产羧酸的最佳微生物细胞工厂。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以权利要求为准。
SEQUENCE LISTING
<110> 天津科技大学
<120> 一种亚细胞区室化生产衣康酸的解脂耶氏酵母工程菌及应用
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tatgtgcagg caacaatgag ctttgagccg ccaggagcct gcagggtgat tggatatgga 240
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tttgcagcaa gtgaggtctt agccgagcaa ggcaaaacaa tttctggtat agatgtcatt 420
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gccatgaagg gtgtcctgga gagatcttat ggcggtttcc tcaaaatgtt caccaagggc 780
aatggcagag agcctcccta caaagaggag gaagtggtgg ccggtctcgg ttcattctgg 840
cataccttta ctattcgcat caagctctat gcctgctgcg gacttgtcca tggtccagtc 900
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gccattgctc agctgacctc tctgggcaag cccctgggct ctctgcatga gtgggtgcac 720
caccaccacc accactaa 738
Claims (7)
1.一种解脂耶氏酵母基因工程菌,其特征在于,所述基因工程菌以解脂耶氏酵母为出发菌株,通过导入顺-乌头酸脱羧酶编码基因获得;所述顺-乌头酸脱羧酶编码基因,其核苷酸如序列表SEQ ID NO.1所示;所述顺-乌头酸脱羧酶编码基因通过增强型过氧化物酶体定位信号ePTS1在过氧化物酶体中进行定位表达;
所述解脂耶氏酵母出发菌株为解脂耶氏酵母(Yarrowia lipolytica)Po1gku70Δ。
2.如权利要求1所述的一种解脂耶氏酵母基因工程菌,其特征在于,所述顺-乌头酸脱羧酶编码基因是通过质粒在宿主细胞中表达。
3.如权利要求2所述的一种解脂耶氏酵母基因工程菌,其特征在于,所述质粒为pYLEX1。
4.如权利要求1所述的一种解脂耶氏酵母基因工程菌,其特征在于,将ePTS1编码基因连接到顺-乌头酸脱羧酶编码基因后,构建重组质粒pYLEX1-CAD-ePTS1,并将重组质粒导入解脂耶氏酵母出发菌株中进行表达。
5.如权利要求1所述的一种解脂耶氏酵母基因工程菌,其特征在于,所述ePTS1编码序列如序列表SEQ ID NO.2所示。
6.权利要求1-5任意一项所述的一种解脂耶氏酵母基因工程菌在生产衣康酸中的应用。
7.如权利要求6所述的应用,采用所述工程菌生产衣康酸的方法如下:
将工程菌种子液按1%接种量接种至YPD培养基中,28-30℃,200-220r/min条件培养15-18h,再转接至YPO培养基中使OD600达到0.1,28-30℃,200-220r/min条件下发酵6天;
所述YPO培养基组成成分为:蛋白胨18-22g/L,酵母浸粉8-12g/L,厨房废油1.1-1.2%,吐温80 0.18-0.22%,余量为水,115-121℃,灭菌20min。
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