CN117363504B - 一种同时生产棕矢车菊素、泽兰林素的酿酒酵母工程菌及其构建方法与应用 - Google Patents
一种同时生产棕矢车菊素、泽兰林素的酿酒酵母工程菌及其构建方法与应用 Download PDFInfo
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- CN117363504B CN117363504B CN202311644882.0A CN202311644882A CN117363504B CN 117363504 B CN117363504 B CN 117363504B CN 202311644882 A CN202311644882 A CN 202311644882A CN 117363504 B CN117363504 B CN 117363504B
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Abstract
本发明涉及一种同时生产棕矢车菊素、泽兰林素的酿酒酵母工程菌及其构建方法与应用。本发明以酿酒酵母株系WAT11为出发菌株,首先构建柚皮素的酵母合成途径,形成EU2;在EU2中整合黄酮合酶FNS和黄酮羟化酶F6H和F3'H的合成基因,构建6‑OH木犀草素的合成途径,形成EU4;在EU4中整合氧甲基转移酶3',4'‑OMT和6‑OMT,形成EU6,即产棕矢车菊素和泽兰林素的酵母细胞。本发明解决了棕矢车菊素和泽兰林素的从酪氨酸起始的异源全合成,为棕矢车菊素和泽兰林素的新药研发提供新的资源获取模式。
Description
技术领域
本发明属于生物合成领域,具体涉及一种同时生产棕矢车菊素、泽兰林素的酿酒酵母工程菌及其构建方法与应用。
背景技术
棕矢车菊素化学名为4',5,7-三羟基-3,6-二甲氧基黄酮;泽兰林素(Eupatilin),又称异泽兰黄素,化学名为5,7- 二羟基 -3',4',6- 三甲氧基黄酮,二者皆是主要存在于艾草等菊科蒿属植物中的黄酮类化合物,具有抗肿瘤、抗炎、抗糖尿病、抗氧化、抗突变、免疫抑制功能等药理活性,具有极高的药用价值,被广泛用于治疗胃炎和消化性溃疡。以棕矢车菊素和泽兰林素为主要活性成分的斯特兰片剂是由东亚制药公司与首尔大学推出的一款新药,具有显著的胃黏膜保护效果。
然而目前棕矢车菊素和泽兰林素的资源获取十分有限,严重限制了其药用价值的开发。艾草等植物中棕矢车菊素和泽兰林素含量低,目前普遍采用的传统提取分离方法不能保证产量,且品质易受气候、地域等因素影响;同时,棕矢车菊素和泽兰林素化学结构较复杂,具有多个甲氧基基团,采用化学合成法选择性低,且在环境、安全和效率等方面也存在诸多问题。因此,如何大量获取棕矢车菊素和泽兰林素以供新药研究和开发是一个亟待解决的问题。
微生物法合成化合物具有反应条件温和、可利用廉价碳源及酶催化特异性强等多重优势。已有研究表明,在大肠杆菌、酵母等微生物异源宿主中引入和表达天然植物途径是一种稳健且可持续的替代方案。目前为止,未见棕矢车菊素和泽兰林素异源生物合成的相关报道。酿酒酵母具有遗传背景清晰、公认安全、生长周期短、便于遗传操作且培养成本低廉等优点。因此,本发明以酿酒酵母作为出发菌株,基于推测的泽兰林素的生物合成路径,利用合成生物性技术,完成棕矢车菊素和泽兰林素全合成途径的构建,具有重要的研究和应用意义。
发明内容
本发明提供一种同时合成棕矢车菊素和泽兰林素的酿酒酵母工程菌,所述酿酒酵母工程菌包括柚皮素的合成途径、6-OH木犀草素的合成途径、棕矢车菊素和泽兰林素的合成途径;所述柚皮素的合成途径为在酿酒酵母株系WAT11上表达FjTAL、Pc4CL、SmCHS2和MsCHI;所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母中表达SbFNSII-1、AtF3'H和SbF6H;所述棕矢车菊素和泽兰林素的合成途径为在6-OH木犀草素合成的酿酒酵母中表达MpalOMT2、ObFOMT4。
本发明的另一实施方案提供上述可同时合成棕矢车菊素和泽兰林素的酿酒酵母工程菌,所述酿酒酵母工程菌的构建方法包括如下步骤:
(1)在酿酒酵母株系WAT11中引入柚皮素的合成途径,得到柚皮素合成的酿酒酵母菌;
(2)在步骤(1)得到的柚皮素合成的酿酒酵母菌中引入6-OH木犀草素的合成途径,得到6-OH木犀草素合成的酿酒酵母菌;
(3)在步骤(2)得到的6-OH木犀草素合成的酿酒酵母菌中表达氧甲基转移酶3',4'-OMT(MpalOMT2)和6-OMT(ObFOMT4),即可引入棕矢车菊素和泽兰林素的合成途径,得到所述酿酒酵母工程菌。
上述技术方案中,步骤(1)所述柚皮素的合成途径为在WAT11上表达FjTAL(酪氨酸解氨酶)、Pc4CL(香豆酰辅酶A连接酶)、SmCHS2(查尔酮合酶)和MsCHI(查尔酮异构酶)。
步骤(2)所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母菌中表达SbFNSII-1(黄酮合酶)、AtF3'H(黄酮3'-羟化酶)和SbF6H(黄酮6-羟化酶)。
本发明的另一实施方案提供一种同时合成棕矢车菊素和泽兰林素的酿酒酵母工程菌的构建方法,包括如下步骤:
(1)在酿酒酵母株系WAT11中引入柚皮素的合成途径,得到柚皮素合成的酿酒酵母菌;
(2)在步骤(1)得到的柚皮素合成的酿酒酵母菌中引入6-OH木犀草素的合成途径,得到6-OH木犀草素合成的酿酒酵母菌;
(3)在步骤(2)得到的6-OH木犀草素合成的酿酒酵母菌中表达氧甲基转移酶3',4'-OMT(MpalOMT2)和6-OMT(ObFOMT4),即可引入棕矢车菊素和泽兰林素的合成途径,得到所述酿酒酵母工程菌。
步骤(1)所述柚皮素的合成途径为在WAT11上表达FjTAL(酪氨酸解氨酶)、Pc4CL(香豆酰辅酶A连接酶)、SmCHS2(查尔酮合酶)和MsCHI(查尔酮异构酶)。
步骤(2)所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母菌中表达SbFNSII-1(黄酮合酶)、AtF3'H(黄酮3'-羟化酶)和SbF6H(黄酮6-羟化酶)。
本发明的另一实施方案提供一种同时合成棕矢车菊素和泽兰林素的酿酒酵母工程菌的构建方法,包括如下步骤:以酿酒酵母株系WAT11为基础菌株,首先构建柚皮素的酵母合成途径,形成EU2;在 EU2中整合黄酮合酶FNS、黄酮羟化酶F6H和F3'H基因的表达盒,构建6-OH木犀草素的合成途径,得到EU4;在EU4中整合氧甲基转移酶3',4'-OMT和6-OMT的表达盒,得到EU6,即产棕矢车菊素和泽兰林素的酿酒酵母工程菌;所述EU2的基因信息为911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do,1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do;所述EU4为在EU2的基础上导入两个基因表达盒:YPRCd15C-Up-pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3’H-pSeGAL2-YPRCd15C-Do和607b-Up-pTDH3-SbF6H-ENO1t-607b-Do;所述EU6为在EU4的基础上导入两个基因表达盒:1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do和416d-Up-tENO1-MpalOMT2-1-pGAL1-416d-Do。
本发明的另一实施方案提供上述酿酒酵母工程菌在制备棕矢车菊素和/或泽兰林素中的应用。
本发明所述FjTAL的DNA序列为SEQ ID NO:1,其包含SEQ ID NO:2的氨基酸序列。
本发明所述Pc4CL的DNA序列为SEQ ID NO:3,其包含SEQ ID NO:4的氨基酸序列。
本发明所述SmCHS2的DNA序列为SEQ ID NO:5,其包含SEQ ID NO:6的氨基酸序列。
本发明所述MsCHI的DNA序列为SEQ ID NO:7,其包含SEQ ID NO:8的氨基酸序列。
本发明所述SbFNSII-1的DNA序列为SEQ ID NO:9,其包含SEQ ID NO:10的氨基酸序列。
本发明所述AtF3'H的DNA序列为SEQ ID NO:11,其包含SEQ ID NO:12的氨基酸序列。
本发明所述SbF6H的DNA序列为SEQ ID NO:13,其包含SEQ ID NO:14的氨基酸序列。
本发明所述MpalOMT2的DNA序列为SEQ ID NO:15,其包含SEQ ID NO:16的氨基酸序列。
本发明所述ObFOMT4的DNA序列为SEQ ID NO:17,其包含SEQ ID NO:18的氨基酸序列。
与现有技术相比,本发明具有以下有益效果:1、本发明在酿酒酵母细胞中构建了同时产棕矢车菊素和泽兰林素的途径,可一步同时产生两种活性黄酮类化合物棕矢车菊素和泽兰林素(韩国上市药物斯特兰片剂的主要活性成分),为棕矢车菊素和泽兰林素的药源获取提供了一种新的方式。2、本发明的酿酒酵母工程菌通过放大优化生产,可大量制备高纯度的棕矢车菊素和泽兰林素,不受环境和土地资源限制。
附图说明
图1是棕矢车菊素和泽兰林素在酿酒酵母工程菌中的生物合成路径图。
图2是柚皮素产物分析图。
图3是6-OH木犀草素产物分析图。
图4是棕矢车菊素和泽兰林素产物分析图。
具体实施方式
以下结合附图和实施例对本中请作进一步详细说明。予以特殊说明的是:以下实施例中未注明具体条件者按照常规条件或制造商建议的条件进行,以下实施例中所用的酶、试剂盒均是可以购买的已经商业化的产品。下述实施例所用的PCR扩增方法、不同片段的融合方法、基因敲除及过表达方法可以采用本领域常见的技术手段,例如融合PCR、同源重组、CRISPR-Cas9技术。
实验方法:
转化,是采用醋酸锂/PEG3350。下述实施例采用的转化方法是:先将宿主菌株在2×YPD培养基中活化,30℃,200 rpm培养过夜。然后接种到新的2×YPD培养基中使起始OD值为0.2,30℃继续培养4-4.5 h后,取5 OD菌液,常温3000 rcf,离心5 min,弃上清液,并用灭菌超纯水洗涤两次,获得酵母细胞;配制DNA混合物,每个构建取5 OD的菌液获得细胞,并与50 μL DNA混合物混合,使细胞重悬,50 μL DNA混合物由2 μg所述插入片段、250 ng工具质粒以及足量ddH2O混合而成。在悬浮的细胞中加入醋酸锂转化混合物,经培养后获取细胞,涂布到筛选平板上,获取单菌落,即为重组酿酒酵母,测序验证转化成功后,将重组酿酒酵母进行保存。
菌落PCR及测序验证:待筛选平板上长出单克隆菌落后,进行菌落PCR及测序验证。具体步骤是:用枪头挑取少量细胞分别置于20 μL的20 mM NaOH溶液,涡旋混匀,于金属浴95℃孵育20 min,涡旋混匀,取1 μL菌液作为模板进行菌落PCR反应,比对克隆条带与阴性克隆条带大小,挑选菌落PCR阳性克隆的菌液送到公司进行测序验证。测序正确的菌株进行划线保存和甘油冻存。
重组酿酒酵母菌株的培养:单菌落置于装有3 mL 1×YPD的24孔板中,在摇床中过夜培养16 h后(条件为30℃,200 rpm),过夜菌液用1×YPD稀释10倍后用紫外分光光度计检测菌液OD,波长设置为600 nm。然后使起始OD为0.2转接至3mL 1×YPG培养液中培养。培养方式为:转接后,每隔24 h添加10 μL酪氨酸(浓度为0.1 M),300 μL的20%半乳糖。培养72 h后收集200 μL菌液为样品。
重组酿酒酵母产物的提取:取样,加入色谱级乙酸乙酯萃取(萃取两次),涡旋1min,12000 rpm离心2 min,吸取上清至洁净的EP管(容量1.5 mL)中,旋干后,在EP管中加入140 μL甲醇,涡旋振荡1 min,离心1 min,进液相小瓶,等待分析。
产物分析和鉴定:产物采用LC-MS分析,仪器型号为 Agilent 1290 Infinity II-6470A,色谱柱型号为:Poroshell 120 EC-C18,2.7 µm,3×100 mm(Agilent),质谱检测模式为负离子全扫模式。色谱分析条件如表1:
表1 色谱条件
时间/time(min) | 流动相 A(0.05%甲酸水) | 流动相 B(0.05%甲酸乙腈) | 流速(mL/min) |
0.00 | 88 | 12 | 0.3 |
5.00 | 75 | 25 | 0.3 |
10.00 | 35 | 65 | 0.3 |
16.00 | 3 | 97 | 0.3 |
19.00 | 3 | 97 | 0.3 |
19.01 | 88 | 12 | 0.3 |
21.00 | 88 | 12 | 0.3 |
主要试剂的配制:
Yeast extract 10 g
Peptone 20 g
D-(+)-Glucose 4 g
加入ddH2O定容至1000 mL,混匀后过滤除菌。
(2)40% D-(+)-Glucose
D-(+)-Glucose 40 g
加入少量ddH2O微波炉加热溶解后,再加ddH2O定容至100 mL,混匀后过滤除菌。
(3)10×YNB溶液
酵母氮源(无氨基酸和硫酸铵)8.5g
硫酸铵25g
加入ddH2O定容至500 mL,过滤除菌。
(4)营养缺陷培养基
首先分别配制SC-Leu-Trp-Ura-His及4种氨基酸的母液(Ura, Trp, His, Leu),配方如下:
5×SC-Leu-Trp-Ura-His | 3.5 g/500 mL |
10× Ura | 0.38 g/ 500 mL |
10×Trp | 0.38 g/ 500 mL |
10×His | 0.38 g/ 500 mL |
10×Leu | 1.8 g/ 500 mL |
然后取上述母液配制SC-Ura型培养基,配方如下:
SC-Ura (100 mL):
5×Sc-leu-trp-ura-his | 20 mL |
10×Trp | 10 mL |
10×Leu | 10 mL |
10×His | 10 mL |
10×(YNB+硫酸铵) | 10 mL |
5%琼脂粉溶液 | 40 mL |
20%葡萄糖 | 10 mL |
(5)醋酸锂转化混合物
50% W/V PEG3350 | 260 uL |
1 M LiOAc | 36 uL |
变性鲑鱼精DNA | 10 uL |
ddH2O | 4 uL |
实施例1 产棕矢车菊素和泽兰林素的酿酒酵母工程菌的构建
一种同时生产棕矢车菊素和泽兰林素的酿酒酵母工程菌,酵母工程菌包括柚皮素的合成途径、6-OH木犀草素的合成途径、棕矢车菊素和泽兰林素的合成途径。本实验选用WAT11酿酒酵母株系。
柚皮素的合成途径为在WAT11上表达FjTAL(酪氨酸解氨酶)、Pc4CL(香豆酰辅酶 A连接酶)、SmCHS2(查尔酮合酶)和MsCHI(查尔酮异构酶)得到柚皮素合成的酿酒酵母菌;其中FjTAL的DNA序列为SEQ IDNO:1其包含SEQ IDNO:2的氨基酸序列;Pc4CL的DNA序列为SEQID NO:3,其包含SEQ ID NO:4的氨基酸序列;SmCHS2的DNA序列为SEQ ID NO:5,其包含SEQID NO:6的氨基酸序列;MsCHI的DNA序列为SEQ ID NO:7,其包含SEQ ID NO:8的氨基酸序列。
6-OH木犀草素的生物合成途径为在柚皮素合成的酿酒酵母中表达SbFNSII-1(黄酮合酶)、 AtF3'H(黄酮3'-羟化酶)和SbF6H(黄酮6-羟化酶)得到6-OH木犀草素合成的酿酒酵母菌;其中SbFNSII-1的DNA序列为SEQ ID NO:9,其包含SEQ ID NO:10的氨基酸序列;AtF3′H的DNA序列为SEQ ID NO:11,其包含SEQ ID NO:12的氨基酸序列;SbF6H的DNA序列为SEQ ID NO:13,其包含SEQ ID NO:14的氨基酸序列。
通过采用上述技术方案,在柚皮素合成的酿酒酵母中表达SbFNSII-1(黄酮合酶)、AtF3'H(黄酮3'-羟化酶)和SbF6H(黄酮6-羟化酶)可以获得6-OH木犀草素的合成途径。
优选地,所述棕矢车菊素和泽兰林素的合成途径为在6-OH木犀草素合成的酿酒酵母中表达氧甲基转移酶3', 4'-OMT(MpalOMT2)和6-OMT(ObFOMT4)。其中MpalOMT2的DNA序列为SEQ ID NO:15,其包含SEQ ID NO:16的氨基酸序列;ObFOMT4的DNA序列为SEQ ID NO:17,其包含SEQ ID NO:18的氨基酸序列。
生产棕矢车菊素和泽兰林素的酿酒酵母工程菌的构建方法,包括以下步骤:
S1、以酿酒酵母株系WAT11为出发菌株,首先构建柚皮素的酵母合成途径,形成EU2:
采用DNA聚合酶进行PCR扩增整合基因片段:以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物Primer1/Primer2(P1/P2,以下引物均采用此缩写方式)扩增911b-UP片段(1000 bp),使用引物P3/P4扩增911b-DOWN片段(1000 bp),得到整合位点上游同源臂911b-UP片段及整合位点下游同源臂911b-DOWN片段;以拥有pSeGAL2-FjTAL-tADH1菌株的基因组为模板,使用引物P5/P6扩增得到pSeGAL2-FjTAL-tADH1片段(2471 bp);以拥有tCYC1-Pc4CL-pTDH3菌株的基因组为模板,使用引物P7/P8扩增得到tCYC1-Pc4CL-pTDH3片段(2438bp)。最终得到911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do表达盒。然后将911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do表达盒片段转化到酿酒酵母WAT11中,得到菌株EU1。
表 2 构建菌株EU1引物序列
引物序号 | 碱基序列 |
1 | SEQ ID NO:19 |
2 | SEQ ID NO:20 |
3 | SEQ ID NO:21 |
4 | SEQ ID NO:22 |
5 | SEQ ID NO:23 |
6 | SEQ ID NO:24 |
7 | SEQ ID NO:25 |
8 | SEQ ID NO:26 |
下一步,继续整合1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do表达盒到EU1基因组中。以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P9/P10扩增1114a-UP片段(1000 bp),使用引物P11/P12扩增1114a-DOWN片段(1000 bp),得到整合位点上游同源臂1114a-UP片段及整合位点下游同源臂1114a-DOWN片段;以拥有ADH1t-SmCHS-pTDH3菌株的基因组为模板,使用引物P13/P14扩增得到ADH1t-SmCHS-pTDH3片段(2020bp);以拥有pSeGal2-MsCHI-TDH1t菌株的基因组为模板,使用引物P15/P16扩增得到pSeGal2-MsCHI-TDH1t片段(1616 bp)。最终得到1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do表达盒。然后将1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do表达盒片段转化到酿酒酵母EU1中,得到菌株EU2。取基因测序正确的菌株进行后续培养。
表3 构建菌株EU2引物序列
引物序号 | 碱基序列 |
9 | SEQ ID NO:27 |
10 | SEQ ID NO:28 |
11 | SEQ ID NO:29 |
12 | SEQ ID NO:30 |
13 | SEQ ID NO:31 |
14 | SEQ ID NO:32 |
15 | SEQ ID NO:33 |
16 | SEQ ID NO:34 |
S2、在EU2中整合黄酮合酶FNS和黄酮羟化酶F6H和F3'H基因的表达盒,构建6-OH木犀草素的合成途径,得到EU4:
以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P17/P18扩增YPRCd15C-Up片段(1000 bp),使用引物P19/P20扩增YPRCd15C-DOWN片段(998 bp),得到整合位点上游同源臂YPRCd-UP片段及整合位点下游同源臂YPRCd-DOWN片段;以包含pSkGAL2-SbFNSII-1-tENO1基因的质粒为模板,使用引物P21/P22扩增得到pSkGAL2-SbFNSII-1-tENO1片段(998 bp);以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P23/P24扩增得到终止子tADH1(250bp);以包含AtF3'H基因的质粒为模板,使用引物P25/P26得到片段AtF3’H(1542 bp);以拥有pSeGAL2启动子菌株的基因组为模板,使用引物P27/P28得到启动子片段pSeGAL2(700bp)。最终得到YPRCd15C-Up- pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3'H-pSeGAL2-YPRCd15C-Do表达盒。然后将YPRCd15C-Up-pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3'H-pSeGAL2-YPRCd15C-Do表达盒片段转化到酿酒酵母EU2中,得到菌株EU3。
表4 构建菌株EU3引物序列
引物序号 | 碱基序列 |
17 | SEQ ID NO:35 |
18 | SEQ ID NO:36 |
19 | SEQ ID NO:37 |
20 | SEQ ID NO:38 |
21 | SEQ ID NO:39 |
22 | SEQ ID NO:40 |
23 | SEQ ID NO:41 |
24 | SEQ ID NO:42 |
25 | SEQ ID NO:43 |
26 | SEQ ID NO:44 |
27 | SEQ ID NO:45 |
28 | SEQ ID NO:46 |
下一步,继续整合607b-Up-pTDH3-SbF6H-ENO1t-607b-Do表达盒到EU3基因组中。以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P29/P30扩增607b-Up片段(1013 bp),使用引物P31/P32扩增607b-DOWN片段(1012 bp),得到整合位点上游同源臂607b-Up片段及整合位点下游同源臂607b-Up片段;以拥有pTDH3启动子菌株的基因组为模板,使用引物P33/P34扩增得到启动子片段pTDH3(600 bp);以包含SbF6H-ENO1t基因的质粒为模板,使用引物P35/P36扩增得到片段SbF6H-ENO1t(1785 bp)。最终得到607b-Up-pTDH3-SbF6H-ENO1t-607b-Do表达盒。然后将607b-Up-pTDH3-SbF6H-ENO1t- 607b-Do表达盒片段转化到酿酒酵母EU3中,得到菌株EU4。取基因测序正确的菌株进行后续培养。
表5 构建菌株EU4 引物序列
引物序号 | 碱基序列 |
29 | SEQ ID NO:47 |
30 | SEQ ID NO:48 |
31 | SEQ ID NO:49 |
32 | SEQ ID NO:50 |
33 | SEQ ID NO:51 |
34 | SEQ ID NO:52 |
35 | SEQ ID NO:53 |
36 | SEQ ID NO:54 |
S3、在酵母细胞EU4上整合氧甲基转移酶3',4'-OMT和6-OMT的表达盒,得到EU6,即产棕矢车菊素和泽兰林素的酿酒酵母工程菌。
以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P37/P38扩增1414a-Up片段(1000 bp),使用引物P39/P40扩增1414a-DOWN片段(1000 bp),得到整合位点上游同源臂1414a-UP片段及整合位点下游同源臂1414a-DOWN片段;以包含tADH1-ObFOMT4的基因组为模板,使用引物P41/P42扩增得到tADH1-ObFOMT4片段(1261 bp);以拥有pSeGAL2启动子菌株的基因组为模板,使用引物P43/P44扩增得到启动子片段pSeGAL2(700 bp)。最终得到1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do表达盒。然后1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do表达盒片段转化到酿酒酵母EU4中,得到菌株EU5。
表6 构建菌株EU5引物序列
引物序号 | 碱基序列 |
37 | SEQ ID NO:55 |
38 | SEQ ID NO:56 |
39 | SEQ ID NO:57 |
40 | SEQ ID NO:58 |
41 | SEQ ID NO:59 |
42 | SEQ ID NO:60 |
43 | SEQ ID NO:61 |
44 | SEQ ID NO:62 |
下一步,以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P45/P46扩增416d-Up片段(1000 bp),使用引物P47/P48扩增416d-DOWN片段(999 bp),得到整合位点上游同源臂416d-UP片段及整合位点下游同源臂416-DOWN片段;以酿酒酵母CEN.PK2-1C的基因组为模板,使用引物P49/P50扩增得到终止子tENO1(225 bp);以包含MpalOMT2-1-pGAL1基因的质粒为模板,使用引物P51/P52扩增得到MpalOMT2-1-pGAL1片段(1521 bp)。最终得到416d-Up-tENO1-MpalOMT2-1-pGAL1-416d-Do表达盒。然后416d-Up-tENO1-MpalOMT2-1-pGAL1-416d-Do表达盒片段转化到酿酒酵母EU5中,得到菌株EU6。取基因测序正确的菌株进行后续培养。
表7 构建菌株EU6引物序列
引物序号 | 碱基序列 |
45 | SEQ ID NO:63 |
46 | SEQ ID NO:64 |
47 | SEQ ID NO:65 |
48 | SEQ ID NO:66 |
49 | SEQ ID NO:67 |
50 | SEQ ID NO:68 |
51 | SEQ ID NO:69 |
52 | SEQ ID NO:70 |
综上,将911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do和1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do两个表达盒转化到酿酒酵母宿主WAT11中,即可在WAT11上构建柚皮素的合成途径,形成酵母细胞EU2。进而将YPRCd15C-Up-pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3’H-pSeGAL2-YPRCd15C-Do和607b-Up-pTDH3-SbF6H-ENO1t-607b-Do两个表达盒转化到酿酒酵母EU2中,即可在EU2上构建6-OH木犀草素的合成途径,形成酵母细胞EU4。最后,将416d-Up- tENO1-MpalOMT2-1-pGAL1-416d-Do和1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do表达盒片段转化到酿酒酵母EU4中,即可构建可同时生产棕矢车菊素和泽兰林素的合成途径,形成酵母菌株EU6。
表8 菌株构建信息
酵母细胞 | 底盘 | 基因信息 |
EU1 | WAT11 | 911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do |
EU2 | EU1 | 1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do |
EU3 | EU2 | YPRCd15C-Up-pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3’H-pSeGAL2-YPRCd15C-Do |
EU4 | EU3 | 607b-Up-pTDH3-SbF6H-ENO1t-607b-Do |
EU5 | EU4 | 1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do |
EU6 | EU5 | 416d-Up-tENO1-MpalOMT2-1-pGAL1-416d-Do |
应用例 一种同时产棕矢车菊素和泽兰林素的酵母细胞的应用
一种同时产棕矢车菊素和泽兰林素的酵母细胞的应用,产棕矢车菊素和泽兰林素的酵母细胞从碳源或酪氨酸生产棕矢车菊素和泽兰林素。
性能检测试验
1、柚皮素产物分析
培养EU2和出发菌株WAT11,收集菌液并进行提取,然后进行LC-MS分析,检测柚皮素的生成。柚皮素在负离子模式下的质/荷比为271。利用柚皮素标准品作为对照,提取质/荷比为271的特征离子峰,可鉴定分析样品中是否含有柚皮素产物。结果如图2所示,出发菌株WAT11不产柚皮素,而表达了柚皮素合成途径的重组菌株EU2检测到了柚皮素的生成。
2、6-OH木犀草素产物分析
培养EU3、EU4和EU2,收集菌液并进行提取,然后进行LC-MS分析,检测木犀草素和6-OH木犀草素的生成。木犀草素和6-OH木犀草素在负离子模式下的质/荷比分别为285和301。提取质/荷比为285和301的特征离子峰,可鉴定分析样品中是否含有木犀草素和6-OH木犀草素产物。结果如图3所示,EU3和EU4中分别检测到了木犀草素和6-OH木犀草素的产物,表明具有6-OH木犀草素合成途径的重组酵母EU4成功生产了6-OH木犀草素。
3、棕矢车菊素和泽兰林素产物分析
培养EU5、EU6和EU4,收集菌液并进行提取,然后进行LC-MS分析,检测6-OMT木犀草素、棕矢车菊素和泽兰林素的生成,负离子模式下三者的质/荷比分别为315、329和343。如图4所示,EU4作为两个氧甲基转移酶的出发菌株,未检测到氧甲基化产物。表达了ObFOMT4的EU5检测到6-OMT木犀草素的生成,进一步表达了MpalOMT2的EU6可同时检测到棕矢车菊素和泽兰林素的生成。
本具体实施例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (5)
1.一种同时合成棕矢车菊素和泽兰林素的酿酒酵母工程菌,其特征在于所述酿酒酵母工程菌包括柚皮素的合成途径、6-OH木犀草素的合成途径、棕矢车菊素和泽兰林素的合成途径;所述柚皮素的合成途径为在酿酒酵母株系WAT11上表达FjTAL、Pc4CL、SmCHS2和MsCHI;所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母中表达SbFNSII-1、AtF3'H和SbF6H;所述棕矢车菊素和泽兰林素的合成途径为在6-OH木犀草素合成的酿酒酵母中表达MpalOMT2、ObFOMT4;
所述FjTAL的DNA序列为SEQ ID NO:1,其包含SEQ ID NO:2的氨基酸序列;
所述Pc4CL的DNA序列为SEQ ID NO:3,其包含SEQ ID NO:4的氨基酸序列;
所述SmCHS2的DNA序列为SEQ ID NO:5,其包含SEQ ID NO:6的氨基酸序列;
所述MsCHI的DNA序列为SEQ ID NO:7,其包含SEQ ID NO:8的氨基酸序列;
所述SbFNSII-1的DNA序列为SEQ ID NO:9,其包含SEQ ID NO:10的氨基酸序列;
所述AtF3'H的DNA序列为SEQ ID NO:11,其包含SEQ ID NO:12的氨基酸序列;
所述SbF6H的DNA序列为SEQ ID NO:13,其包含SEQ ID NO:14的氨基酸序列;
所述MpalOMT2的DNA序列为SEQ ID NO:15,其包含SEQ ID NO:16的氨基酸序列;
所述ObFOMT4的DNA序列为SEQ ID NO:17,其包含SEQ ID NO:18的氨基酸序列。
2.根据权利要求1所述的酿酒酵母工程菌,其特征在于所述酿酒酵母工程菌的构建方法包括如下步骤:
(1)在酿酒酵母株系WAT11中引入柚皮素的合成途径,得到柚皮素合成的酿酒酵母菌;
(2)在步骤(1)得到的柚皮素合成的酿酒酵母菌中引入6-OH木犀草素的合成途径,得到6-OH木犀草素合成的酿酒酵母菌;
(3)在步骤(2)得到的6-OH木犀草素合成的酿酒酵母菌中表达氧甲基转移酶MpalOMT2和ObFOMT4,即可引入棕矢车菊素和泽兰林素的合成途径,得到所述酿酒酵母工程菌;
步骤(1)中所述柚皮素的合成途径为在酿酒酵母株系WAT11上表达FjTAL、Pc4CL、SmCHS2和MsCHI;
步骤(2)中所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母中表达SbFNSII-1、AtF3'H和SbF6H。
3.一种权利要求1所述酿酒酵母工程菌的构建方法,其特征在于包括如下步骤:
(1)在酿酒酵母株系WAT11中引入柚皮素的合成途径,得到柚皮素合成的酿酒酵母菌;
(2)在步骤(1)得到的柚皮素合成的酿酒酵母菌中引入6-OH木犀草素的合成途径,得到6-OH木犀草素合成的酿酒酵母菌;
(3)在步骤(2)得到的6-OH木犀草素合成的酿酒酵母菌中表达氧甲基转移酶MpalOMT2和ObFOMT4,即可引入棕矢车菊素和泽兰林素的合成途径,得到所述酿酒酵母工程菌;
步骤(1)中所述柚皮素的合成途径为在酿酒酵母株系WAT11上表达FjTAL、Pc4CL、SmCHS2和MsCHI;
步骤(2)中所述6-OH木犀草素的合成途径为在柚皮素合成的酿酒酵母中表达SbFNSII-1、 AtF3'H和SbF6H。
4.根据权利要求3所述的构建方法,其特征在于包括如下步骤:以酿酒酵母株系WAT11为基础菌株,首先构建柚皮素的酵母合成途径,形成EU2;在EU2中整合黄酮合酶FNS、黄酮羟化酶F6H和F3'H基因的表达盒,构建6-OH木犀草素的合成途径,得到EU4;在EU4中整合氧甲基转移酶3',4'-OMT和6-OMT的表达盒,得到EU6,即产棕矢车菊素和泽兰林素的酿酒酵母工程菌;所述EU2的基因信息为911b-Up-pSeGAL2-FjTAL-tADH1/tCYC1-Pc4CL-pTDH3-911b-Do,1114a-Up-ADH1t-SmCHS-pTDH3-pSeGal2-MsCHI-TDH1t-1114a-Do;所述EU4为在EU2的基础上导入两个基因表达盒:YPRCd15C-Up-pSkGAL2-SbFNSII-1-tENO1-tADH1-AtF3’H-pSeGAL2-YPRCd15C-Do和607b-Up-pTDH3-SbF6H-ENO1t-607b-Do;所述EU6为在EU4的基础上导入两个基因表达盒:1414a-Up-tADH1-ObFOMT4-pSeGAL2-1414a-Do和416d-Up-tENO1-MpalOMT2-1-pGAL1-416d-Do。
5.权利要求1-2任一项所述的酿酒酵母工程菌在制备棕矢车菊素和/或泽兰林素中的应用。
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