CN113383073A - 二羧酸合成相关酶及使用其生产二羧酸的方法 - Google Patents
二羧酸合成相关酶及使用其生产二羧酸的方法 Download PDFInfo
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- CN113383073A CN113383073A CN201980091206.0A CN201980091206A CN113383073A CN 113383073 A CN113383073 A CN 113383073A CN 201980091206 A CN201980091206 A CN 201980091206A CN 113383073 A CN113383073 A CN 113383073A
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Abstract
本发明涉及二羧酸合成相关酶、编码该酶的基因以及使用该酶生产二羧酸的方法。本发明的基因或由该基因编码的酶代替现有的二羧酸化学生产,可用于生物酶法生产二羧酸,因此预期具有高的工业实用性。
Description
技术领域
本发明涉及一种参与二羧酸(DCA)生产的酶、编码该酶的基因、包含该基因的载体以及使用该酶生产二羧酸的方法。
背景技术
二羧酸(Dicarboxylic acid,DCA)是含有两个羧基基团(-COOH)的有机化合物。二羧酸的分子通式可以用HO2C-R-CO2H来表示,其中R可以是脂肪族或芳香族基团。通常,二羧酸表现出类似于单羧酸的化学反应和反应性。二羧酸也用于制备共聚物,例如聚酰胺和聚酯。工业中使用最广泛的二羧酸是己二酸,它是用于生产尼龙的前体。二羧酸的其他例子包括天冬氨酸和谷氨酸,它们是人体中的两种氨基酸。此外,其他羧酸已用于各种工业领域。
此类二羧酸已通过化学工艺或生物方法制备。作为关于制备二羧酸的一个例子,作为二羧酸中的一种的癸二酸,即使使用苯酚和甲酚也可以合成它,但已知蓖麻油氧化是最环保且价格最具竞争力的方法。蓖麻油通过蒸汽裂解进行酯交换,并通过酯交换生产蓖麻油酸。将如此产生的蓖麻油酸在250℃下加热后与碱(例如熔融苛性钠等)混合,经苛性碱消化分解为辛醇(2-辛醇)和癸二酸。将如此产生的产物纯化以得到高纯度癸二酸(美国专利第5,952,517和6,392,074号)。然而,这种方法的缺点是需要在300℃或更高的高温处理下才能达到上述目的,并且使用诸如硫酸的强酸,以及由于使用了诸如重金属、有毒有机溶剂等物质而产生大量的环境污染物。除了使用化学方法制备癸二酸外,还可以通过电解己二酸单乙酯钾(Potassium monoethyl adipate)来进行这种生产。
在之前的研究中,已经报道了使用ω-氧化能力优异且β-氧化被阻断的热带假丝酵母(Candida tropicalis)菌株通过生物方式来生产二羧酸。然而,没有关于具体二羧酸生物合成途径的报道。因此,确定二羧酸生物合成途径以开发能够大量生产二羧酸的有用菌株是重要的。
因此,本发明人已经发现通过使用产生二羧酸的热带假丝酵母菌株的进化方法来筛选与二羧酸生物合成相关的基因,并且使用这些基因来确定生物合成途径。因此,基于这些事实完成了本发明。
[相关技术文件]
[专利文件]
韩国专利申请第10-2015-0149253号
[非专利文件]
David L.Craft等人,《应用与环境微生物学》,69(10):5983-5991,2003
发明内容
技术问题
因此,本发明的一个目的是提供一种参与二羧酸(DCA)生物合成的蛋白质,该蛋白质包括选自以下中的一个或多个:脂肪酶(LIP1)、细胞色素P450 52B1(CYP52B1)、NADPH-细胞色素P450还原酶(NCP1)、长链醇氧化酶(FAO1)和醛脱氢酶(ALD1)。
本发明的另一个目的是提供包含编码该蛋白质的基因的重组载体,以及包含该重组载体的用于生产二羧酸的组合物。
本发明的又一个目的是提供一种用于生产二羧酸的方法,该方法包括培养用包含该基因的载体转化的微生物。
技术方案
在以前的研究中,二羧酸的生产是通过化学方法进行的,并且大多数已知生产二羧酸的菌株都由于易受到底物的细胞毒性的影响而死亡。因此,通过生物方法生产二羧酸或确定二羧酸生产途径并不容易。于是,本发明人使用先前研究中的进化方法制造了对细胞毒性底物具有高耐受性并且产生二羧酸的菌株,并从该菌株中筛选了与二羧酸生物合成相关的酶和编码该酶的基因。因此,基于这些事实完成了本发明。
特别地,本发明提供一种参与二羧酸(DCA)生物合成的蛋白质,该蛋白质包括选自以下蛋白质中的一个或多个:脂肪酶(LIP1)、细胞色素P450 52B1(CYP52B1)、NADPH-细胞色素P450还原酶(NCP1)、长链醇氧化酶(FAO1)和醛脱氢酶(ALD1)。
该蛋白质可以源自热带假丝酵母菌株,但本发明不特别限于此。
根据一个实施方式,热带假丝酵母菌株已知是产生作为二羧酸的一种的癸二酸的菌株,将该热带假丝酵母菌株在含有表现出细胞毒性的底物的培养基中培养以筛选在进化方面具有优异的在底物中存活能力的菌株,以及通过对筛选的菌株进行基因组分析,选择推测与二羧酸代谢相关的菌株的内源基因,即分别由SEQ ID NO:1-5所示的碱基序列表示的脂肪酶基因、细胞色素P450 52B1(CYP52B1)基因、NADPH-细胞色素P450还原酶(NCP1)基因、长链醇氧化酶基因和醛脱氢酶基因。结果证实,由该基因表达的酶在该酶在体外与底物发生酶促反应产生二羧酸。
脂肪酶可以由SEQ ID NO:1所示的基因表达;细胞色素P450 52B1(CYP52B1)可以由SEQ ID NO:2所示的基因表达;NADPH-细胞色素P450还原酶(NCP1)可以由SEQ ID NO:3所示的基因表达;长链醇氧化酶可以由SEQ ID NO:4所示的基因表达;以及醛脱氢酶可以由SEQ ID NO:5所示的基因表达。
此外,分别由SEQ ID NO:1-5所示的碱基序列表示的基因是包括一个或多个突变的基因,所述突变例如取代、缺失、易位、添加等。在这种情况下,每种由基因表达的酶还包括具有脂肪酶、细胞色素P450 52B1、NADPH-细胞色素P450还原酶、长链醇氧化酶和醛脱氢酶的酶活性的基因。特别地,每种酶包括与SEQ ID NO:1-5所示的碱基序列之一的序列同源性为80%或更多、85%或更多、90%或更多、93%或更多、94%或更多、95%或更多、96%或更多、97%或更多、98%或更多以及99%或更多的碱基序列。
一个或多个基因可以被包括在载体中。载体可以是基因可以可操作连接的形式。在本发明中,术语“可操作连接”一般意指将碱基表达调控序列与编码所需蛋白质的碱基序列可操作连接以执行其功能,从而对编码所需蛋白质的碱基序列的表达产生影响。可以使用本领域已知的基因重组技术来实现载体的可操作连接,并且可以使用本领域已知的消化酶和连接酶等进行位点特异性DNA消化和连接。
在本发明中,术语“载体”是指任何用于将碱基克隆和/或转移到宿主细胞中的培养基。载体可以是可以与另一个DNA片段结合以复制结合片段的复制子。术语“复制子”是指任何在体内作为DNA复制的自体单位起作用、也就是说可通过其自身调节进行复制的遗传单位(例如,质粒、噬菌体、粘粒、染色体、病毒)。术语“载体”可以包括用于在体外、离体或体内将碱基引入宿主细胞的病毒介质和非病毒介质。此外,术语“载体”可以包括微球形DNA。例如,载体可以是不具有细菌DNA序列的质粒。术语“载体”还可以包括转座子,例如Sleeping Beauty(Izsvak等人.J.MoI.Biol.302:93-102(2000)),或人工染色体。常用载体的例子包括天然存在的或重组的质粒、粘粒、病毒和噬菌体。例如,pWE15、M13、MBL3、MBL4、IXII、ASHII、APII、t10、t11、Charon4A、Charon21A等可以用作噬菌体载体或粘粒载体。也可以使用质粒载体。本发明中可以使用的载体没有特别限制,可以使用公知的表达载体。优选地,可以使用过表达基因的载体。
本发明提供了一种用于生产二羧酸的组合物,该组合物包含包括上述五种蛋白质中的一种或多种蛋白质,本发明还提供了一种用于生产二羧酸的组合物,该组合物包含重组载体,该重组载体包含分别编码蛋白质的SEQ ID NO:1-5所示的一个或多个基因。
本发明提供包含一个或多个基因的重组载体,以及用包含该重组载体的组合物转化的具有生产二羧酸能力的微生物。
例如,微生物可以是藻类、病毒、细菌、酵母和真菌。更特别地,微生物可以是热带假丝酵母菌株。热带假丝酵母菌株是一种β-氧化途径被阻断的菌株。特别地,热带假丝酵母菌株是β-氧化途径被阻断从而使用底物产生二羧酸的菌株。
此外,本发明提供了一种用于生产二羧酸的方法,该方法包括将选自以下中的一种或多种蛋白质与底物一起培养:脂肪酶(LIP1)、细胞色素P450 52B1(CYP52B1)、NADPH-细胞色素P450还原酶(NCP1)、长链醇氧化酶(FAO1)和醛脱氢酶(ALD1)。
根据本发明的具体实施方式,该方法可以是包括以下步骤的用于生产二羧酸的方法:
(1)使脂肪酶(LIP1)与C6-C20脂肪酸甲酯进行酶促反应;
(2)使步骤(1)的产物与细胞色素P450 52B1(CYP52B1)和NADPH-细胞色素P450还原酶(NCP1)进行酶促反应;
(3)使步骤(2)的产物与长链醇氧化酶(FAO1)进行酶促反应;以及
(4)使步骤(3)的产物与醛脱氢酶(ALD1)进行酶促反应。
根据本发明的酶促生产二羧酸的方法可以在体外进行,并且时序性的酶促反应步骤可以视为二羧酸生物合成的新途径。
再根据另一方面,本发明提供了一种用于生产二羧酸(DCA)的方法,该方法包括将用包含编码蛋白质的基因的载体转化的微生物与底物一起培养。
根据本发明的用于生产二羧酸(DCA)的方法原样利用上述脂肪酶(LIP1)、细胞色素P45052B1(CYP52B1)、NADPH-细胞色素P450还原酶(NCP1)、长链醇氧化酶(FAO1)和醛脱氢酶(ALD1)或编码这些蛋白质的基因。因此,为避免本说明书过于复杂,省略对该蛋白质和该基因之间的共同内容的描述。
用于生产二羧酸的方法中使用的底物可以是脂肪酸甲酯(Fatty acid methylester,FAME)。特别地,脂肪酸甲酯可以是包括C6-C20亚烷基基团的脂肪酸甲酯中的一种。更特别地,脂肪酸甲酯可以是癸酸甲酯(Decanoic acid methyl ester,DAME)。
用包含所述基因的载体转化的微生物不受限制,但热带假丝酵母菌株可以优选是β-氧化途径被阻断的菌株。
有益效果
已经证实,根据本发明获得的基因与二羧酸的产生有关。此外,已经发现由这些基因表达的酶表现出产生二羧酸前体物质的活性。因此,这适用于酶促地或以生物方式生产二羧酸的工艺,克服了现有二羧酸化学生产工艺的弊端,更加环保、安全,因此预期具有高的工业实用性。
附图说明
图1是示出了作为二羧酸的一种的癸二酸的生物合成途径以及与该生物合成途径相关的基因的示意图;
图2示出了Lip1p酶的体外反应产物的GC/MS分析结果;
图3示出了Cyp52B1p酶和Ncp1p酶的体外反应产物的GC/MS分析结果;
图4示出了Fao1p酶和Ald1p酶的体外反应产物的GC/MS分析结果。
具体实施方式
下面其结合实施方式对本发明的构成和效果进行更详细的说明。但是,应当理解,本文所描述的实施方式仅用于举例说明本发明,并不意在限制本发明的范围。
[实施例1]使用进化工程方法开发耐DAME菌株
为了开发对具有细胞毒性的底物的DAME具有耐受性的菌株,将热带假丝酵母(C.tropicalis)MYA_3404菌株在添加了10g/L浓度的DAME的YNB培养基(10g/L酵母提取物和20g/L蛋白胨)中培养。在这种情况下,证实了由于DAME底物的低溶解度,培养基中DAME的浓度保持在大约0.45g/L(最大溶解度)(通过内部实验的结果证实)。接种菌株的生长曲线通过测量600nm波长处的吸光度值来确定。
实时观察接种了菌株的培养基的吸光度,然后将菌株在新鲜培养基中传代培养,直至菌株生长达到中期指数期。由测得的吸光度值计算出菌株的比生长速率,将比生长速率变化很大的阶段的菌株分别确定为E1(170代时)、E2(470代时)、E3(650代时)、E4(700代时间)和E5(720代时)。此外,将通过上述方法获得的E5菌株在补充有20g/L葡萄糖作为无毒碳源的YNB培养基(10g/L酵母提取物和20g/L蛋白胨)中进行传代培养,然后在DAME底物中重新接种以筛选即使在更换碳源后仍保持对DAME耐受性的菌株,将该菌株命名为“ES5”。
[实施例2]DAME耐受突变株(ES5)的转录组分析
为了考察含有和不含DAME的培养基中转录组的变化,对在补充有DAME的培养基中生长的ES5菌株和在不含DAME的培养基中生长的ES5菌株的转录组进行了分析。
将ES5菌株在不含DAME的YNB培养基和补充有10g/LDAME的YNB培养基中于30℃培养24小时。收集培养的细胞,并用水洗涤。之后,将收集的细胞用作用于全RNA提取的样品。使用RNeasy Mini Kit(Qiagen,Hilden,Germany)进行RNA提取,并分别使用NanoDrop(Thermo Scientific,Wilmington,DE,USA)和Agilent Bioanalyzer 2100(Santa Clara,Ca,USA)来测量提取的RNA的浓度和纯度。
对突变型ES5菌株的转录组进行分析,并与亲本菌株的转录组进行比较。结果证实,与亲本菌株相比,在ES5菌株中总共有453个基因上调,并且与亲本菌株相比,ES5菌株中有147个基因下调。表1中详述了基因的数量和聚类的详细信息。在通过转录组分析确认上调的453个基因中,选择了预计与作为二羧酸中的一种的癸二酸的代谢相关的五个基因(LIP1、CYP52B1、NCP1、FAO1和ALD1)(图1)。
[表1]
亲本菌株和DAME耐受突变株(ES5)的转录组的比较/分析结果
[实施例3]利用克隆技术获得SA生物合成途径相关基因
基于实施例2的转录组分析结果,选择了预期与癸二酸生物合成途径相关的5个基因(LIP1、CYP52B1、NCP1、FAO1和ALD1),通过克隆获得LIP1(Uniprot.ID:C5MD87)、CYP52B1(Uniprot.ID:C5MAM3)、NCP1(Uniprot.ID:C5M346)、NADPH-细胞色素P450还原酶、FAO1(Uniprot.ID:Q6QIR6)和ALD1(Uniprot.ID:C5M346)基因,以检查源自5个基因的酶(脂肪酶、细胞色素P450 52B1、长链醇氧化酶和醛脱氢酶)的活性。已知CYP450基因有两个亚基,CYP1和NCP1。
将热带假丝酵母MYA_3404菌株在YPD培养基(10g/L酵母提取物、20g/L蛋白胨和20g/L葡萄糖)中于30℃培养48小时,然后使用酵母DNA分离试剂盒(Epicentre,Madison,WI,USA)提取用于克隆的模板DNA。使用Q5 High-Fidelity Master mix(BioLabs,Ipswich,MA,USA)扩增候选基因,扩增候选基因所用的引物列于表2(引物1至10;SEQ ID NO:6-15)。之后,所有基因重组实验均使用相同的酶进行PCR。添加编码组氨酸残基的基因的碱基序列以增强HisTrap柱的亲和力。将除CYP450基因之外的其余PCR产物和pAUR123载体用XhoI和XbaI限制酶双酶切消化,并使用T4 DNA连接酶(New England Biolabs)将最终DNA片段连接到相同的限制酶切位点。作为编码CYP450基因的两个亚基,CYP52B1(Uniprot.ID:C5MAM3)和NCP1(Uniprot.ID:C5M346)基因分别用SmaI/SalI、SalI/XhoI消化,然后依次连接。之后,将所有四种连接的质粒(质粒1至4)各自转化到大肠杆菌(E.coli)DH5α(Novagen,Cambridge,MA,USA)中。为了过表达由通过上述过程获得的基因编码的蛋白质,将四种提取的质粒(质粒1至4)再次转化到热带假丝酵母菌株中。根据LiAc/SS载体DNA/PEG方法使用酵母转化试剂盒(MPBiomedicals,Solon,OH,USA)来进行对酵母的转化。通过用作基因导入载体的pAUR123载体的自动诱导系统,蛋白质在没有任何单独诱导剂的情况下自发表达。
[表2]
用于克隆SA生物合成相关基因的引物列表
限制醇切位点用下划线表示
[表3]
[实施例4]癸二酸生物合成途径相关酶的过表达及纯化
为了使在实施例3中获得的重组菌株中的癸二酸生物合成相关酶过表达,将重组菌株在补充有0.2mg/L的金担子素A的YPD培养基(10g/L酵母提取物、20g/L蛋白胨和20g/L葡萄糖)中于30℃培养24小时。为了分离表达的蛋白质,超声破碎细胞并离心。然后,使用HisTrap柱(GE Healthcare,Piscataway,USA)纯化上清液。使用Amicon UltraCentrifugal过滤器(Millipore,Billerica,MA,USA)浓缩纯化的蛋白质。证实了表达的酶的分子量为50.6kDa(Lip1p)、59.3kDa(Cyp1)和76.7kDa(Ncp1)(Cyp450p)、77.8kDa(Fao1p)、以及61.3kDa(Ald1p),如通过SDS-PAGE测量的。使用二辛可宁酸(Bicinchoninicacid,BCA)蛋白质测定试剂盒(Pierce,Rockford,IL,USA)测量蛋白质的浓度。
[实施例5]癸二酸生物合成途径相关酶的体外活性的确认
为了考察实施例4中获得的Lip1p、Cyp52B1p、Ncp1、Fao1p和Ald1p的活性,使用每种底物相应的标准物质进行体外酶测定。
为了考察Lip1p的活性,使50μL的100mM DAME底物、250μL的酶(酶浓度:5mg/mL)和200μL的20mM Tris-HCl反应,即总共500μL的反应样品在30℃反应1小时。DAME与失活的Lip1p酶反应,所得反应产物用作对照。如下图2所示,GC/MS结果显示出DAME的峰降低,而通过Lip1p酶与底物反应得到的反应产物中产生了新的峰。然后,将该峰的质谱与标准物质的质谱进行比较。结果,证实了新产生的峰对应于DA。
为了使用与上述相同的方法考察Cyp52B1p和Ncp1p的活性,使50μL的100mM DA底物、100μL的酶(酶浓度:2.99mg/ml)和50μL的20mM Tris-HCl反应,即总共200μL的反应样品在30℃反应1小时。DA与灭活的Cyp52B1p酶和Ncp1p酶反应,所得反应产物用作对照。如下图3所示,GC/MS结果显示出,DA的峰降低,而通过Cyp52B1p酶和Ncp1p酶与底物反应得到的反应产物中产生了新的峰。然后,证实了该峰的质谱。结果,证实了新产生的峰对应于10-羟基癸酸(图3)。
因为没有购买预期为反应产物的标准物质(10-氧代癸酸),因此Fao1p酶的活性测定如下。即,10μL的100mM 10-HAD作为底物与作为最终的生物合成相关酶的100μL Fao1p(酶浓度:2.7mg/ml)和100μL Ald1p(酶浓度:2.0mg/ml)依次反应,然后分析反应产物。结果,证实产生了SA(图4)。
根据上述结果,确定了与作为二羧酸中的一种的癸二酸的生物合成相关的酶的活性,并且可以基于这些结果预测二羧酸的新生物合成途径。
序列表
<110> 高丽大学校产学协力团
<120> 二羧酸合成相关酶及使用其生产二羧酸的方法
<130> G21U13C0487P/CN
<150> KR 10-2018-0154373
<151> 2018-12-04
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 1398
<212> DNA
<213> Candida tropicalis
<400> 1
atgcaattgt caattgtttg cttctttctt ttaacatatg ttaatgctgc tgtcatcaca 60
ttgacaaaac catcggatga tgatttctac attccacaag atggttttga agatgctgaa 120
ccaggtgaga ttttgaagat ccgaaagacc cccaacaagc ttagtggttt atttttccca 180
atagatgtca aaaatagttg gcaattgtta gtacgctcta ctgattcctt tggaaatgcc 240
actgctattg ttgctactat catagaacca ttcaattctg atccttccaa agtggtatcc 300
tatcaaactt ttgaagattc ggccaatatt aactgttcac cttcctatgg gatgcaatgg 360
ggtgcttcta tttccactgt tgcgacacag attgatatgt catttatggt cccattgtta 420
aacaatggtt actttttggt atctccagat tatgaaggtc ctaaatcaac tttcactgtg 480
ggtagacaat cgggacatgg tactttggac tcaatcagag ccattttaca atctggaaat 540
tttactggtg ttgatgaaga tgcacaggtt gctatgtggg gttattctgg tggttctttg 600
gcatcaggtt gggctgctgc tttgcaacca cattatgcac ctgaattgga agataatttg 660
attggtgctg cccttggtgg atttgtaacc aatattacag ctactgctga agctacagat 720
ggaaagttat tggctggctt agtacctatt gctttgaatg gattgggtaa tgaatataat 780
gatttccgtg aaatattgta ctctgaagtc aaagaaggtg gaagagataa attggctgat 840
gggttaaatc attgtatgat tcctggtatc attcgctttg cctttagcca atttctagca 900
ggtaaaaata gactattccc taatggttac ggtttgctcg acgacccaat agttaacaga 960
acaatccagg aaaacaatct tatgagtgtc tccaaggagt acattcctaa aataccgtta 1020
tttgtttatc atggcacatt ggatgcagtg gttccaattg tcaatgtcaa gaaaacctat 1080
gaaagatggt gtgattgggg tattgaatct tttgaatttg ctgaagactt gttgaatggt 1140
cacatttctg aaactttagt tggtgcacca gctgcgttga catggttgga aagaagattc 1200
gctggtttgg atccagtcaa aggatgccaa catactgcac gaatgatgaa cttcttatac 1260
ccaaatatct caagtgctac tagtgattac tttacaggat tgtatgatgc tcttcaaggc 1320
accccattgg gaccaggatt gaacaccgac aactttacac ttaatggatt gctaggaacc 1380
ttgggtgata ttttctag 1398
<210> 2
<211> 1554
<212> DNA
<213> Candida tropicalis
<400> 2
atgatcgaac aagttgttga atactggtac gtggtcttac cattggtatt tatccttcat 60
aaagtatttg acatgtggca cactcgtcgg ttgatgaagc aattgggcgc tgctcctgtc 120
acaaaccaat tacacgacaa tttttttggt attatcaacg gatggaaagc acttaagttc 180
aagaaagaag gtagagctca agaatataat gattataaat ttgccaattc gaaaattcca 240
agtgtgggta cttatgttag taccatcttt ggaacaaagc tcctcgtcac aaaagatccg 300
gagaatatca aagctttatt agcaacccaa ttcagtgatt tttccttggg taagaggcat 360
acacttttca aaccattatt aggtgacggt attttcactt tggatggaga aggttggaaa 420
catagtcgag ctatgttgag accacagttt gcaagagaac aagttgccca tgtcacttct 480
ttagagccac atttccaatt gttgaaaaaa catatcctca agaacaaagg aggttttttt 540
gatatccagg aattgttttt ccgattcact gttgattcag ctactgagtt tttgtttggt 600
gaatcagtac actctttgaa ggatgaaaca attgggtata accaagatga tatcgacttt 660
gttggtagaa aggattttgc ggaatcgttc aacaaggcac aagagtatct tgctattaga 720
actttggtgc aagattttta ttatcttgta aacaatcagg aattcagaga ttgcaataaa 780
ctggtacaca agtttaccaa ctactatgtc caaagagcat tggatgccac tcctgaagag 840
cttgaaaagc aaagtggata tgttttcttg tatgaattgg ttaaacaaac cagagaccct 900
aatgtattga gagatcaatc attgaacatc ttattagctg gtagagatac cactgctggg 960
ttgttgtcat ttgcggtatt tgaacttgct aggaatccac atatttgggc caaattaaga 1020
gaagatgttg aatcccaatt tggtcttggt gaagaatctc gcattgaaga gattaccttt 1080
gaaagtttaa aacgatgtga atacttgaaa gctttcctta acgaaacatt acgtgtttat 1140
ccaagtgttc caagaaattt cagaattgct accaaaaaca ccactttacc aagaggtggt 1200
ggttcagacg gcaattctcc tgttttggtc aaaaagggcg aggctgtttc atatggtata 1260
aattctactc acttagatcc tgtctattat ggtgacgatg ctgcagaatt tagaccagaa 1320
agatggaacg agccatcaac aagaaaattg ggatgggcat atttaccgtt caacggaggc 1380
ccaagaattt gtttaggtca acaatttgct ttaaccgaag cgggttatgt attggttaga 1440
ttggcccaaa gttttgacac cttggaattg aagccaccag ttgtgtatcc accaaagaga 1500
ttaacaaact tgactatgtc tttacaagac ggaactattg tcaagatcga ttag 1554
<210> 3
<211> 2043
<212> DNA
<213> Candida tropicalis
<400> 3
atggcattag ataagttaga tttatatgtt attataacat tggtggttgc aattgcagct 60
tattttgcaa agaatcagtt tcttgaccaa caacaagata ccgggttcct taatactgat 120
agtggagatg gtaattcaag agatatctta caagctttga agaagaacaa taaaaatacg 180
ttattattat ttggatccca aacaggtaca gcagaagatt atgccaacaa attgtcaaga 240
gaattgcatt caagatttgg tttgaaaacc atggttgctg atttcgctga ttatgatttc 300
gaaaacttcg gagatattac tgaagatatc ttggttttct ttattgttgc tacttatggt 360
gaaggtgaac caaccgataa tgctgacgaa tttcacactt ggttgactga agaagctgac 420
accttgagta ctttgaaata tactgttttt ggtttgggta attcaactta tgaattcttc 480
aatgctattg gtagaaaatt tgacagattg ttgggagaaa aaggtggtga cagatttgct 540
gaatacggtg aaggtgacga tggtactggt actttagatg aagatttctt ggcctggaag 600
gataacgtgt ttgattcctt aaagaatgat ttgaattttg aagaaaaaga gttgaaatac 660
gaaccaaatg ttaaattgac tgaaagagat gatttatctg gcaatgatcc agaagtctcc 720
ttgggtgaac caaatgtcaa atacattaaa tctgaaggtg ttgacttaac taaaggtcca 780
tttgatcata ctcatccatt tttggctaga attgttaaaa ctaaagaatt gtttacttct 840
gaagacagac attgtgttca tgttgaattt gatatttctg aatcaaactt gaaatatacc 900
accggtgatc atcttgcaat ctggccatct aactctgatg aaaacattaa gcaatttgcc 960
aaatgttttg gtttagaaga caaacttgat actgttattg aattgaaagc tttggattcc 1020
acttattcca tcccattccc taatccaatc acttatggag ctgttattag acaccatttg 1080
gaaatttcag gtcctgtttc tagacaattt ttcttatcta ttgctggatt tgcccctgat 1140
gaagaaacta aaaagtcatt tactagaatt ggtggtgata agcaagaatt tgctagtaaa 1200
gtcacccgta gaaaattcaa cattgccgat gctttattat ttgcttccaa caacagacca 1260
tggtccgatg ttccattcga attccttatt gaaaatgtcc aacacttaac tcctcgttat 1320
tactccattt cttcttcctc attaagtgaa aagcaaacca ttaatgttac tgctgttgtt 1380
gaagccgaag aagaagctga tggaagacca gttactggtg ttgtcaccaa cttgttgaag 1440
aatattgaaa ttgaacaaaa caaaactggt gaaaccccaa tggttcatta tgatttgaat 1500
ggtccaagag gcaaatttag caagttcaga ttgccagttc acgttagaag atctaatttc 1560
aaattaccaa agaatagcac taccccagtt attttgattg gtccaggtac cggtgttgca 1620
ccattgagag gttttgttag agaaagagtt caacaagtta aaaatggtgt taatgttggt 1680
aagactgtat tgttttatgg atgtagaaat tccgaacaag atttcttgta caaacaagaa 1740
tggagtgaat atgcctcagt attgggagaa aatttcgaaa tgtttaatgc cttctcaaga 1800
caagatccaa ctaagaaagt ttatgttcaa gataagattt tagaaaatag tgctcttgtt 1860
gatgagttat tatctagtgg agcaattatt tatgtttgtg gtgatgccag tagaatggct 1920
agagatgttc aagctgcaat tgccaagatt gttgccaaaa gtagagatat ccacgaagat 1980
aaagctgctg aattggttaa atcttggaaa gttcaaaata gataccaaga agatgtctgg 2040
taa 2043
<210> 4
<211> 4296
<212> DNA
<213> Candida tropicalis
<400> 4
tgcatactcg gagcatatcg ccatcgtcca tatcgttggc actccatcca ctgagtcagc 60
caagaagcaa ttgttgttgc accacacctt aggtaatggt gactttactg ttttccacaa 120
gatctcgtca ttcatcagtg ccactactgc tgggttgacc gacccagaca ccgccgctga 180
tgaaattgat agagtgattg agtcagccta catcaaccag cgtccaacgt acttgggatt 240
cccttccaac atggttgacg ttcaagtgcc agtcagcaag ttggacaagc cattgaactt 300
aaccccacct gcaaacaatc caaagatcca gtctgaggtc ttgagcgaca ttattgcctt 360
gattgaaacc gccaaggatc cagttatcat cattgatgct tgttgtggaa ggcacaatgc 420
taccccagag gcacagaagt tgattgagtt gacaaagttc aagtttgctg tcaccccaat 480
ggctaaaggg tctaaggaca ttgatgaaag tgatccgaag ttcattggtt gctacgttgg 540
tgacttgtct tatccaagag tcaaagagtt ggttgaaagc tcggacttgg tcttgtcctt 600
gggtgctgtc ttgtctgatt tcaacactgg ttcgttctca tactctttgg acaatgccaa 660
ggttgttgaa ttccactccg actacactca aatcaagagc gctcagtacc caggtatcag 720
aatgaaggaa ttgttgggca agttggttga ggagccagaa ttggtcaaga cgtgttccaa 780
gatcccagca aagaagttgg tcactgacaa ctttgaacca ttggtcttgc caccggacca 840
caagctcacc caatcctggt tgtggagtaa cttgggtaat tggttgaaag aaggtgatgt 900
gattgttacc gaaaccggta cttccaattt cggtattgtc cagaccaaat tcccaaagaa 960
tgctgtcggt atctcgcaag tcttgtgggg ttccattggc tactcggtcg gttctgccgc 1020
tggtgccgtt atcgccgccg aggagcttga tcccagccgt agagtcatct tgtttgttgg 1080
tgacggttct ttgcagttga ccgtgcagga aatctccacc atggccagac acaagaacaa 1140
catctacatc tttgtcttga acaacaacgg tttcaccatt gaaagattga ttcacggtcc 1200
agaagctggt tacaacagta ttcaagaatg ggagaacgct gagttattga agactttcaa 1260
ggctaccaac tacgagagtt tcaccgtcaa gactgtcggc gaacttgaca aggtgttcaa 1320
ggatgaaaag tttgccgtca acgacaagat tagattggtt gagatcatgt tagacacttt 1380
cgatgctcca gagaacttgg ttaagcaagc tgagagatct gccaacacca acaagtagag 1440
tttgtctatg ttttccgttt gccttttctt tctagtacga gacgttattg aacgaagttt 1500
ttatatatct agatctaata catattccat gtctgttcat ttttgacgga gtttcataag 1560
gtggcagttt ctaatcaaag gtccgtcatt ggcgtcgtgg cattggcggc tcgcatcaac 1620
tcgtatgtca atattttctg ttaactccgc cagacatacg atcaaaacct acaagcaaaa 1680
aaattccaca tgctttgttt gagatctcca caaacaacaa cggggtaaga aaatcatggg 1740
gcgattaatc atgccatctt tgtaaatttc tttgtttcaa catcaccctc tttagtcaaa 1800
ccttcacagg actgtctgct ctactttgcc acccagttca tatataaatt accaacttcc 1860
accgagcacc accaacacct caccccactc tctccccccc cctttttttt ccagcttaga 1920
cacacacttc aaactcgaca tggctccatt tttgcccgac caggtcgact acaaacacgt 1980
cgacaccctt atgttattat gtgacgggat catccacgaa accaccgtgg acgaaatcaa 2040
agacgtcatt gcccctgact tccccgccga caaatacgag gagtacgtca ggacattcac 2100
caaaccctcc gaaaccccag ggttcaggga aaccgtctac aacaccgtca acgcaaacac 2160
catggatgca atccaccagt tcattatctt gaccaatgtt ttgggatcaa gggtcttggc 2220
accagctttg accaactcgt tgactcctat caaggacatg agcttggaag accgtgaaaa 2280
gttgttagcc tcgtggcgtg actcccctat tgctgctaaa aggaagttgt tcaggttggt 2340
ttctacgctt accttggtca cgttcacgag attggccaat gagttgcatt tgaaagccat 2400
tcattatcca ggaagagaag accgtgaaaa ggcttatgaa acccaggaga ttgacccttt 2460
taagtaccag tttttggaaa aaccgaagtt ttacggcgct gagttgtact tgccagatat 2520
tgatgtgatc attattggat ctggggccgg tgctggtgtc gtggcccaca ctttgaccaa 2580
cgacggcttc aagagtttgg ttttggaaaa gggcagatac tttagcaact ccgagttgaa 2640
ctttgatgac aaggacgggg ttcaagaatt ataccaaagt ggaggtactt tgaccaccgt 2700
caaccagcag ttgtttgttc ttgctggttc cacttttggt ggtggtacca ctgtcaattg 2760
gtcggcctgt cttaaaacgc cattcaaggt gcgtaaggaa tggtatgatg agtttggcgt 2820
tgactttgct gccgatgaag cctacgacaa agcacaggat tatgtttggc agcaaatggg 2880
agcttctacc gaaggcatca cccactcttt ggctaacgag attattattg aaggtggcaa 2940
gaaattaggt tacaaggcca aggtattaga ccaaaacagc ggtggtcatc ctcatcacag 3000
atgcggtttc tgttatttgg gttgtaagca cggtatcaag cagggctctg ttaataactg 3060
gtttagagac gcagctgccc acggttctca gttcatgcaa caggttagag ttttgcaaat 3120
ccttaacaag aagggcatcg cttatggtat cttgtgtgag gatgttgtaa ccggtgccaa 3180
gttcaccatt actggcccca aaaagtttgt tgttgccgcc ggcgccttaa acactccatc 3240
tgtgttggtc aactccggat tcaagaacaa gaacatcggt aagaacttaa ctttgcatcc 3300
agtttctgtc gtgtttggtg attttggcaa agacgttcaa gcagatcact tccacaactc 3360
catcatgact gctctttgtt cagaagccgc tgatttagac ggcaagggtc atggatgcag 3420
aattgaaacc atcttgaacg ctccattcat ccaggcttca ttcttaccat ggagaggtag 3480
taacgaggct agacgagact tgttgcgtta caacaacatg gtggccatgt tacttcttag 3540
tcgtgatacc accagtggtt ccgtttcgtc ccatccaact aaacctgaag cattagttgt 3600
cgagtacgac gtgaacaagt ttgacagaaa ctccatcttg caggcattgt tggtcactgc 3660
tgacttgttg tacattcaag gtgccaagag aatccttagt ccccaaccat gggtgccaat 3720
ttttgaatcc gacaagccaa aggataagag atcaatcaag gacgaggact atgtcgaatg 3780
gagagccaag gttgccaaga ttccttttga cacctacggc tcgccttatg gttcggcgca 3840
tcaaatgtct tcttgtcgta tgtcaggtaa gggtcctaaa tacggtgctg ttgataccga 3900
tggtagattg tttgaatgtt cgaatgttta tgttgctgac gctagtcttt tgccaactgc 3960
tagcggtgct aatcctatgg tcaccaccat gactcttgca agacatgttg cgttaggttt 4020
ggcagactcc ttgaagacca aggccaagtt gtagttctgt atacgtatct tataatttag 4080
atttcctttt attgacggta aacattcagg ataggtacta cccttgctgc aaaagcccag 4140
cacgccccaa tcgcgatgac ttgagcgaag caaacacgca cacaaaaggg gtacacaaaa 4200
aataacgaga tgcccttgaa gcacacaccc aaacacgatg gaacacaaga tggccctaga 4260
aagtacaaaa aaagtaaagc cacttgattc cgccca 4296
<210> 5
<211> 1653
<212> DNA
<213> Candida tropicalis
<400> 5
atgacaccac cttctaaaat tgaggacagt tcatcatcat catcctcatc agcagattct 60
tccacttttg ttactggtaa ttcatggtat actaaagtat cagatattaa accaggtgtt 120
gatagattaa ttgacgcttt tcatactaaa caaaagactc aagatgttca atttcgttta 180
aatcaattga gaaaccttta ttatgccgtt caagataata ctgatgaatt atgtgaagct 240
ttagagaaag attttcatcg tgcaaatact gaaactaaaa atttagaaat tgttggtgga 300
ttagctgaat tagttcatac tatgtcaagt ttacacgaat ggttgaaacc tgaaaaagtt 360
actgatttac caattacttt aaaaactaat ccaatttata ttgaaagaat tccattaggt 420
gttgttttaa ttataagtcc atttaattat cctttctttt tatcattttc tgctattgtt 480
ggtgcaattg ctggtggtaa tgcagttgtt ttaaaacaat ctgaattagc tcctaatttt 540
tcaagtttat ttagtaaaat tttaactaat gctttagatc cagatatttt cttttctgtt 600
gatggtggaa ttcctgaaat tactgaatta ttagaacaaa aatttgataa aattatgtat 660
actggtaata atactgttgg taaaattatt gctaaaaaag ccgctgaaac tttaactcca 720
gttattttag aattaggagg aaaatcacct gcttttattt tagatgatgt taaagataaa 780
gatttagaag ttattgctag aagaattgca tggggtagat ttaccaatgc tggtcaaact 840
tgtgttgctg ttgattatgt tttagtttgt gaaaaacttc ataaaaaatt tgttgatatt 900
ttaattaaaa ttttacatga agaattttat cctggtttaa ataaagatac taaaggttat 960
actcatatta ttcatgatcg agcatttaac aatttatcaa agattatttc aaccacaaaa 1020
ggagatgttg tatttggagg agaaacagat gctaattcaa gatttatttc tccaactgtt 1080
attgataatg catcatggga tgattcatca atgaaaggag aaatttttgg tccaatttta 1140
ccaattttat catatgataa attatcagat gctattaaac aagttttaaa tcaacatgat 1200
actccattag ctcaatatat tttcacatct ggttcaactt cacgtaaata taatcgtcaa 1260
ttggatcaaa ttttaactta tgttagatca ggtggggtta ttatcaatga tgttttaatg 1320
catgttgctt taattaatgc tccatttggt ggtattggta attctggtta tggttcttat 1380
catggtaaat tttcttttag aagttttact catgaacgta ctacaatgga acaaaaatta 1440
tggaatgata atatggttaa agtaagatat cctccttata attcaaataa agataaattg 1500
attaaaattt ctcaacaaaa tttcaatggt aaagtttggt ttgatagaaa tggtgatgtt 1560
ccagttaatg gaccaagtgg gttatttact acatggactt caattactgg tgtctttagt 1620
ttacttgctg aattcataac caataaacaa taa 1653
<210> 6
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> LIP1_F
<400> 6
aaactcgaga tgagatttct tgtattcatt acaattatta catggttgaa aac 53
<210> 7
<211> 61
<212> DNA
<213> Artificial Sequence
<220>
<223> LIP1_R
<400> 7
aaatctagag tggtggtggt ggtggtggac aagataggta ctattcttca cagtgaagct 60
t 61
<210> 8
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> CYP52B1_F
<400> 8
aaacccggga tgatcgaaca agttgttgaa tactggtacg tg 42
<210> 9
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> CYP52B1_R
<400> 9
aaagtcgacg tggtggtggt ggtggtgatc gatcttgaca atagttccgt cttgtaaaga 60
ca 62
<210> 10
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> NCP1_F
<400> 10
aaagtcgaca tggcattaga taagttagat ttatatgtta ttataacatt ggtg 54
<210> 11
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> NCP1_R
<400> 11
aaactcgagg tggtggtggt ggtggtgcca gacatcttct tggtatctat tttgaacttt 60
cc 62
<210> 12
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> FAO1_F
<400> 12
aaactcgaga tggctccatt tttgcccgac caggt 35
<210> 13
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> FAO1_R
<400> 13
aaatctagag tggtggtggt ggtggtgcaa cttggccttg gtcttcaagg agtct 55
<210> 14
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> ALD1_F
<400> 14
aaactcgaga tgacaccacc ttctaaaatt gaggacagtt ca 42
<210> 15
<211> 61
<212> DNA
<213> Artificial Sequence
<220>
<223> ALD1_R
<400> 15
aaatctagag tggtggtggt ggtggtggac aagataggta ctattcttca cagtgaagct 60
t 61
Claims (15)
1.一种参与二羧酸(DCA)生物合成的蛋白质,所述蛋白质包含选自以下中的一个或多个:脂肪酶(LIP1)、细胞色素P450 52B1(CYP52B1)、NADPH-细胞色素P450还原酶(NCP1)、长链醇氧化酶(FAO1)和醛脱氢酶(ALD1)。
2.根据权利要求1所述的蛋白质,其中,所述蛋白质源自热带假丝酵母(Candidatropicalis)菌株。
3.根据权利要求1所述的蛋白质,其中,所述二羧酸是C6-C20二羧酸。
4.根据权利要求1至3中任一项所述的蛋白质,其中,所述脂肪酶(LIP1)由SEQ ID NO:1所示的基因表达;所述细胞色素P450 52B1(CYP52B1)由SEQ ID NO:2所示的基因表达;所述NADPH-细胞色素P450还原酶(NCP1)由SEQ ID NO:3所示的基因表达;所述长链醇氧化酶(FAO1)由SEQ ID NO:4所示的基因表达;以及所述醛脱氢酶(ALD1)由SEQ ID NO:5所示的基因表达。
5.一种用于二羧酸生物合成的组合物,所述组合物包含重组载体,所述重组载体包含选自由以下基因组成的组中的一个或多个基因:SEQ ID NO:1所示的基因;SEQ ID NO:2所示的基因;SEQ ID NO:3所示的基因;SEQ ID NO:4所示的基因;以及SEQ ID NO:5所示的基因。
6.一种具有生产二羧酸(DCA)的能力的微生物,其中所述微生物用权利要求5中定义的组合物转化。
7.根据权利要求6所述的微生物,其中,所述微生物是β-氧化途径被阻断的热带假丝酵母菌株。
8.一种用于生产二羧酸(DCA)的方法,所述方法包括:
将权利要求1至4中任一项所定义的蛋白质与底物一起培养。
9.根据权利要求8所述的方法,其中,所述底物是脂肪酸甲酯(FAME)。
10.根据权利要求9所述的方法,其中,所述脂肪酸甲酯底物包括选自C6-C20脂肪酸甲酯中的一种或多种。
11.根据权利要求8所述的方法,其中,所述方法包括:
(1)使脂肪酶(LIP1)与C6-C20脂肪酸甲酯进行酶促反应;
(2)使步骤(1)的产物与细胞色素P450 52B1(CYP52B1)和NADPH-细胞色素P450还原酶(NCP1)反应;
(3)使步骤(2)的产物与长链醇氧化酶(FAO1)进行酶促反应;以及
(4)使步骤(3)的产物与醛脱氢酶(ALD1)进行酶促反应。
12.一种用于生产二羧酸(DCA)的方法,所述方法包括:
将权利要求6中定义的微生物与底物在培养基中培养。
13.根据权利要求12所述的方法,其中,所述微生物是β-氧化途径被阻断的热带假丝酵母菌株。
14.根据权利要求12所述的方法,其中,所述底物是脂肪酸甲酯(FAME)。
15.根据权利要求14所述的方法,其中,所述脂肪酸甲酯包括选自C6-C20脂肪酸甲酯中的一种或多种。
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KR1020180154373A KR102173101B1 (ko) | 2018-12-04 | 2018-12-04 | 디카르복시산 생합성 관련 효소 및 이를 이용한 디카르복시산 생산방법 |
KR10-2018-0154373 | 2018-12-04 | ||
PCT/KR2019/017017 WO2020116932A2 (ko) | 2018-12-04 | 2019-12-04 | 디카르복시산 생합성 관련 효소 및 이를 이용한 디카르복시산 생산방법 |
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CN107532186A (zh) * | 2015-03-27 | 2018-01-02 | 油料作物与高蛋白作物领域发展基金会 | 微生物和其用于生产二酸的用途 |
CN108473993A (zh) * | 2015-10-27 | 2018-08-31 | 韩国生命工学研究院 | 中链氨基羧酸的生产方法 |
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US20020061566A1 (en) * | 2000-03-20 | 2002-05-23 | Eirich L. Dudley | Biooxidation capabilities of candida sp |
US8158391B2 (en) * | 2009-05-06 | 2012-04-17 | Dna Twopointo, Inc. | Production of an α-carboxyl-ω-hydroxy fatty acid using a genetically modified Candida strain |
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CN107532186A (zh) * | 2015-03-27 | 2018-01-02 | 油料作物与高蛋白作物领域发展基金会 | 微生物和其用于生产二酸的用途 |
CN108473993A (zh) * | 2015-10-27 | 2018-08-31 | 韩国生命工学研究院 | 中链氨基羧酸的生产方法 |
Non-Patent Citations (7)
Title |
---|
"GenBank accession no. AY538780.1", NCBI * |
"GenBank accession no. XM_002545658.1", NCBI * |
"GenBank accession no. XM_002548772.1", NCBI * |
"Genbank Accession no. XM_002549845.1", NCBI * |
"GenBank accession no. XM_002550127.1", NCBI * |
"GenBank accession no. XP_002550173.1" * |
LEE ET AL.: "Development of a promising microbial platform for the production of dicarboxylic acids from biorenewable resources", BIOTECHNOLOGY FOR BIOFUELS, no. 11, pages 310 * |
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WO2020116932A3 (ko) | 2021-04-22 |
WO2020116932A2 (ko) | 2020-06-11 |
KR20200068144A (ko) | 2020-06-15 |
US20220049231A1 (en) | 2022-02-17 |
KR102173101B1 (ko) | 2020-11-03 |
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