CN114958637B - 一种产β-桉叶醇工程菌及其构建方法、应用 - Google Patents
一种产β-桉叶醇工程菌及其构建方法、应用 Download PDFInfo
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Abstract
本申请适用于微生物技术领域,提供了一种产β‑桉叶醇工程菌及其构建方法、应用。所述产β‑桉叶醇工程菌以酿酒酵母为出发菌株,过表达tHMGR、UPC2‑1、ASG1和ZSS2基因,敲低ERG9基因;其中,tHMGR和UPC2‑1基因整合到酿酒酵母染色体TY3位点;ERG9基因的启动子替换为半乳糖抑制型启动子HXT1p;ASG1基因的启动子替换为组成型强启动子GAL1p;ZSS2基因整合到酿酒酵母染色体的TY1位点。将本申请提供的产β‑桉叶醇工程菌经活化后接种到发酵培养基中进行发酵培养,可得到高产量的β‑桉叶醇,实现利用合成生物学的手段高产量制备β‑桉叶醇的方法。
Description
技术领域
本申请属于微生物技术领域,尤其涉及一种产β-桉叶醇工程菌及其构建方法、应用。
背景技术
β-桉叶醇是中药苍术的主要药效成分,具有抗肿瘤、保护神经中枢、保护肠胃、利尿等广泛的药理活性。此外,β-桉叶醇在啤酒花中含量较高,是啤酒辛辣风味来源之一,广泛用作食品添加剂,具有很高的经济价值。
目前,β-桉叶醇的获得主要有两种方式,一是从苍术中直接提取,但苍术为虫媒植物,单性花多为雌花,种子发芽率、座果率较低,根茎增长缓慢,旺盛的市场需求导致对苍术的肆意挖掘,造成道地性药材的资源破坏;二是化合合成的方法,但合成过程中使用大量的有机溶剂,成本高且影响环境,商业价值过低。
近年来,合成生物学在规模化生产重要的中药活性成分(尤其是植物源天然产物)方面取得了突破性进展。很多中药成分,如青蒿素、灯盏乙素、人参皂苷、丹参酮等已经通过合成生物学手段构建了相应的工程菌,为规模化生产奠定了重要基础。虽然β-桉叶醇的生源合成途径已经得到解析,很多β-桉叶醇合成酶基因从药用植物中被鉴定到,但至今尚缺乏利用合成生物学手段生产β-桉叶醇的微生物细胞工厂。
发明内容
本申请的目的在于提供一种产β-桉叶醇工程菌,旨在填补利用合成生物学手段生产β-桉叶醇的微生物细胞工厂的技术空白。
本申请是这样实现的,一种产β-桉叶醇工程菌,所述产β-桉叶醇工程菌以酿酒酵母为出发菌株,过表达tHMGR、UPC2-1、ASG1和ZSS2基因,敲低ERG9基因;其中,
tHMGR和UPC2-1基因整合到酿酒酵母染色体TY3位点;
ERG9基因的启动子替换为半乳糖抑制型启动子HXT1p;
ASG1基因的启动子替换为组成型强启动子GAL1p;
ZSS2基因整合到酿酒酵母染色体的TY1位点;
所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1。
本申请的另一目的在于一种产β-桉叶醇工程菌的构建方法,包括如下步骤:
将GAL1p、tHMGR和ADH1t连接,构建基因表达模块I;
将GAL10p、UPC2-1和CYC1t连接,构建基因表达模块II;所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1;
将所述基因表达模块I和基因表达模块II连接,构建基因表达模块III;
将筛选标记MET和启动子HXT1p连接,构建基因表达模块IV;
将筛选标记LEU和启动子GAL10p连接,构建基因表达模块V;
将GAL10p、ZSS2和CYC1t连接,构建基因表达模块VI;
将基因表达模块III插入到载体pCfB2875的SfaSI酶切位点,得到整合表达载体pCfB2875-III;
用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2875-III,得到DNA整合片段S1;
将所述DNA整合片段S1整合到酿酒酵母染色体的TY3位点,得到菌株ZZ01;
将基因表达模块IV转化所述菌株ZZ01,得到菌株ZZ02;
将基因表达模块V转化所述菌株ZZ02,得到菌株ZZ03;
将基因表达模块VI插入到载体pCfB2988的SfaSI酶切位点,得到整合表达载体pCfB2988-VI;
用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2988-VI,得到DNA整合片段S2;
将所述DNA整合片段S2整合到酿酒酵母染色体的TY1位点,得到产β-桉叶醇工程菌。
本申请的另一目的在于一种上述的产β-桉叶醇工程菌或者根据上述的产β-桉叶醇工程菌的构建方法所构建得到的产β-桉叶醇工程菌在生产β-桉叶醇中的应用。
本申请的另一目的在于一种β-桉叶醇的生产方法,包括:
将上述的产β-桉叶醇工程菌或者根据上述的产β-桉叶醇工程菌的构建方法所构建得到的产β-桉叶醇工程菌经活化后接种到发酵培养基中进行发酵培养,得到β-桉叶醇。
本申请提供的产β-桉叶醇工程菌,该菌株的出发菌株为酿酒酵母菌株,其构建过程包括,过表达tHMGR、UPC2-1和ASG1基因,以及一个苍术中β-桉叶醇合成酶编码基因经密码子优化而来的ZSS2基因,同时抑制ERG9基因的表达。将本申请提供的产β-桉叶醇工程菌经活化后接种到发酵培养基中进行发酵培养,可得到高产量的β-桉叶醇,实现利用合成生物学的手段高产量制备β-桉叶醇的方法。
附图说明
图1为本申请实施例所构建的β-桉叶醇酿酒酵母菌株的构建示意图;
图2为本申请实施例提供的发酵产物β-桉叶醇的GC-MS检测结果。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本申请提供了一种产β-桉叶醇工程菌,为菌株ZZ04。
所述菌株ZZ04以酿酒酵母为出发菌株,过表达tHMGR、UPC2-1、ASG1和ZSS2基因,敲低ERG9基因;其中,
tHMGR和UPC2-1基因整合到酿酒酵母染色体TY3位点;
ERG9基因的启动子替换为半乳糖抑制型启动子HXT1p;
ASG1基因的启动子替换为组成型强启动子GAL1p;
ZSS2基因整合到酿酒酵母染色体的TY1位点;
可选地,所述酿酒酵母优选为酿酒酵母BY4741。
所述tHMGR和UPC2基因可从酿酒酵母BY4741基因组中克隆得到,其核苷酸序列分别如SEQ ID NO.1~2所示。
所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1,其核苷酸序列如SEQ ID NO.3所示。
所述ZSS2基因为苍术中的β-桉叶醇合成酶经密码子优化而来,其核苷酸序列如SEQ ID NO.4所示。
所述启动子HXT1p和GAL1p可从酿酒酵母BY4741基因组克隆得到,其核苷酸序列如SEQ ID NO.5~6所示。
具体地,所述产β-桉叶醇工程菌的构建方法,包括如下步骤:
(1)利用重叠PCR构建以下模块:
(a)将GAL1p、tHMGR和ADH1t连接,构建基因表达模块GAL1p-tHMGR-ADH1t,命名为模块I;
(b)将GAL10p、UPC2-1和CYC1t连接,构建基因表达模块GAL10p-UPC2-1-CYC1t,命名为模块II;
(c)将模块I和模块II连接,构建基因表达模块GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t,命名为模块III;
(d)将筛选标记MET和启动子HXT1p连接,构建基因表达模块MET-HXT1p,命名为模块IV;
(e)将筛选标记LEU和启动子GAL10p连接,构建基因表达模块LEU-GAL10p,命名为模块V;
(f)将GAL10p、ZSS2和CYC1t连接,构建基因表达模块GAL10p-ZSS2-CYC1t,命名为模块VI;
(2)构建菌株ZZ01
将步骤(1)中得到的模块III插入到载体pCfB2875的SfaSI酶切位点,得到整合表达载体pCfB2875-III;然后用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2875-III,得到DNA整合片段S1;再将DNA整合片段S1整合到菌株BY4741染色体的TY3位点,得到菌株ZZ01。
(3)构建菌株ZZ02
将步骤(1)中得到的模块IV转化酵母工程菌ZZ01,得到菌株ZZ02。
(4)构建菌株ZZ03
将步骤(1)中得到的模块V转化酵母工程菌ZZ02,得到菌株ZZ03。
(5)构建菌株ZZ04
将步骤(1)中得到的模块VI插入到载体pCfB2988的SfaSI酶切位点,得到整合表达载体pCfB2988-VI;然后用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2988-VI,得到DNA整合片段S2;再将DNA整合片段S2整合到菌株BY4741染色体的TY1位点,得到菌株ZZ04。
(6)将步骤(5)中得到的菌株ZZ04发酵,所得到的化合物即为β-桉叶醇。
步骤(1)中所述的tHMGR和UPC2基因可从酿酒酵母BY4741基因组克隆得到,其核苷酸序列分别如SEQ ID NO.1~2所示。
步骤(1)中所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1,其核苷酸序列如SEQ ID NO.3所示。
步骤(1)中所述的ZSS2基因为苍术中的β-桉叶醇合成酶经密码子优化而来,其核苷酸序列如SEQ ID NO.4所示。
步骤(1)中所述启动子HXT1p、GAL1p和GAL10p可从酿酒酵母BY4741基因组克隆得到,其核苷酸序列如SEQ ID NO.5~7所示。
步骤(1)中所述终止子ADH1t和CYC1t可从酿酒酵母BY4741基因组克隆得到,其核苷酸序列如SEQ ID NO.8~9所示。
步骤(1)中所述的筛选标记MET和LEU的核苷酸序列如SEQ ID NO.10~11所示。
可选地,步骤(2)~(5)中所述的酵母菌株优选菌株为BY4741。
可选地,步骤(2)~(5)中所述的转化为采用酵母转化试剂盒进行转化;优选为用Zymo Research Frozen-EZ Yeast Transformation II KitTM 酵母转化试剂盒进行转化。
步骤(6)所述的产物为β-桉叶醇(C15H26O),其结构式如下所示:
。
具体地,所述产β-桉叶醇工程菌的构建步骤,如图1所示,过表达甲羟戊酸途径中的限速酶基因tHMGR和转录因子基因UPC2-1,过表达促进脂肪油滴和脂肪酸降解途径的调控因子基因ASG1,替换鲨烯合酶基因ERG9的启动子为半乳糖抑制型启动子HXT1p以抑制三萜合成途径,过表达β-桉叶醇合酶基因ZSS2;其中,tHMGR和UPC2-1基因整合到酿酒酵母染色体TY3位点;ERG9基因的启动子替换为半乳糖抑制型启动子HXT1p;ASG1基因的启动子替换为组成型强启动子GAL1p;ZSS2基因整合到酿酒酵母染色体的TY1位点。
本申请还提供了一种生产β-桉叶醇的方法,为将所述的生产β-桉叶醇的酿酒酵母工程菌经活化后接种到发酵培养基中进行发酵培养,得到β-桉叶醇;具体包括如下步骤:
将所述的β-桉叶醇的酿酒酵母工程菌进行活化,然后接种到发酵培养基中进行发酵培养,得到β-桉叶醇。
可选地,所述活化为多级活化,通过以下操作实现:挑取所述的β-桉叶醇的酿酒酵母工程菌单克隆,接种到5 mL的YPD培养基中,30℃条件下220 rpm震荡培养至OD值为2~3;然后接种到15 mL的YPD培养基中,30℃条件下220 rpm震荡培养至OD值为2~3;再将菌液接种到100 mL发酵培养基中,30℃条件下220 rpm震荡培养至OD值为8~10。
其中,所述发酵培养基的成分如下:磷酸二氢钾(KH2PO4) 8 g/L,硫酸铵((NH4)2SO4) 15 g/L,硫酸镁(MgSO4) 3 g/L,七水硫酸锌(ZnSO4•7H2O) 0.72 g/L,维生素溶液12mL/L,微量金属盐溶液10 mL/L,葡萄 25 g/L;其中:
可选地,维生素溶液的成分为:肌醇25 g/L,盐酸硫胺素1 g/L,盐酸吡哆醇1 g/L,烟酸1 g/L,泛酸钙1 g/L,维生素H 0.05 g/L和对氨基苯甲酸0.2 g/L。
可选地,微量金属盐溶液的成分为:乙二胺四乙酸(EDTA) 15 g/L,七水硫酸锌(ZnSO4•7H2O) 10.2 g/L,四水氯化锰(MnCl2•4H2O) 0.5 g/L,硫酸铜(CuSO4) 0.5 g/L,六水氯化钴(CoCl2•6H2O) 0.86 g/L,二水钼酸钠(Na2MoO4•2H2O) 0.56 g/L,二水氯化钙(CaCl2•2H2O) 3.84 g/L和七水硫酸铁(FeSO4•7H2O) 5.12 g/L。
其中,所述补料培养基组成如下:葡萄糖 585 g/L,磷酸二氢钾(KH2PO4)9 g/L,硫酸钾(K2SO4) 3.5 g/L,硫酸镁(MgSO4) 2.5 g/L,硫酸钠(Na2SO4) 0.28 g/L,维生素溶液 12mL/L,以及微量金属盐溶液10 mL/L;其中:
可选地,维生素溶液:肌醇25 g/L,盐酸硫胺素1 g/L,盐酸吡哆醇1 g/L,烟酸1 g/L,泛酸钙1 g/L,维生素H 0.05 g/L和对氨基苯甲酸0.2 g/L。
可选地,微量金属盐溶液:乙二胺四乙酸(EDTA) 15 g/L,七水硫酸锌(ZnSO4•7H2O)10.2 g/L,四水氯化锰(MnCl2•4H2O) 0.5 g/L,硫酸铜(CuSO4) 0.5 g/L,六水氯化钴(CoCl2•6H2O) 0.86 g/L,二水钼酸钠(Na2MoO4•2H2O) 0.56 g/L,二水氯化钙(CaCl2•2H2O)3.84 g/L和七水硫酸铁(FeSO4•7H2O) 5.12 g/L。
所述的发酵培养的条件优选为:温度30℃,转速300~1000 rpm,通气量3~20 L/min,溶氧值30%, pH 5.0,发酵时间144 h。
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明,本发明所用试剂和原材料均可通过市售获得。
实施例1 酵母基因和表达元件的克隆
1、酵母基因组DNA的提取
(1)将酿酒酵母BY4741的单克隆挑取至含有5 mL YPD培养基的10 mL试管中,30℃培养过夜,然后4000 rpm离心2 min进行集菌。
(2)弃上清,将菌体转移至液氮预冷的研钵中。
(3)加入液氮,研磨,待液氮挥干后,将粉末转移至1.5 mL离心管中。
(4)利用基因组DNA纯化试剂DNAiso Reagent(9770Q,宝日医生物技术有限公司)进行基因组DNA的提取,具体操作过程见说明书。
2、酵母基因和表达元件的克隆
(1)以上述获得的酵母基因组DNA为模板,分别克隆以下两个个基因、两个启动子和两个终止子(扩增引物见表1):
基因tHMGR(引物为tHMGR-F和tHMGR-R)、基因UPC2(引物为UPC2-F和UPC2-R)、启动子HXT1p(引物为HXT1p-F和HXT1p-R)、启动子GAL1p(引物为GAL1p-F和GAL1p-R)、启动子GAL10p(引物为GAL10p-F和GAL10p-R)、终止子CYC1t(引物为CYC1t-F、CYC1t-R)和终止子ADH1(引物为ADH1t-F、ADH1t-R)。
PCR反应体系:KAPA Taq ReadyMix(KK1006,默克) 10 μL,上下游引物各0.5 μL,ddH2O 7 μl,模板1 μL,总反应体系为20 μL。
PCR扩增反应条件为:98℃ 1 min;98℃ 30 s,55-60℃ 30 s,72℃ 1-2 min,32循环;72℃ 10 min。
PCR反应结束后,利用1%琼脂糖凝胶电泳检测目的条带大小,并使用琼脂糖凝胶DNA回收试剂盒(DP219,天根生化科技有限公司)回收目的条带,具体操作过程见说明书。
(2)DNA片段连接亚克隆载体
将回收的目的DNA片段连接平末端亚克隆载体pEASY-Blunt(CB101,全式金生物技术有限公司),具体操作见产品说明书,并转化感受态大肠杆菌DH5α,涂布平板。
(3)将平板置于37℃培养箱中,倒置培养过夜。
(4)挑选单菌落做菌落PCR验证目的基因与亚克隆载体的连接,PCR反应体系如下:Easy Taq聚合酶0.2 μL(AP111,全式金生物技术有限公司),dNTPs(2.5 mM)0.8 μL,10 xEasy Taq Buffer 1 μL,上下游引物各0.3 μL,ddH2O 7.4 μL。
PCR扩增条件为:95℃ 3 min;95℃ 30 s,55-60℃ 30 s,72℃ 1-2 min,30个循环;72℃ 7 min。
PCR反应结束后,琼脂糖凝胶电泳检测条带大小,挑取阳性克隆菌株测序。
表1 实施例中克隆各基因和表达元件所用的引物序列
引物名称 | 引物序列(5’-3’) |
tHMGR-F | ATGGCTGCAGACCAATTGGTGAA |
tHMGR-R | TTAGGATTTAATGCAGGTGACGGACC |
UPC2-F | ATGAGCGAAGTCGGTATACAGAATCACA |
UPC2-R | TCATAACGAAAAATCAGAGAAATTTGTTGTTG |
UPC2-1-F | GAATACAGTGGAGGTGGTGATATGCATATGATGCTAGA |
UPC2-1-R | TCTAGCATCATATGCATATCACCACCTCCACTGTATTC |
ZSS2-F | ATGGAAAAGCAGTCTTTGACCTTCGA |
ZSS2-R | TTACTTGTTAAAGTAATCGCAGGATTCAAC |
HXT1p-F | TGCAGGTCTCATCTGGAATATAATTCC |
HXT1p-R | GATTTTACGTATATCAACTAGTTGACGATTATGA |
GAL1p-F | AGTACGGATTAGAAGCCGCCG |
GAL1p-R | GGGTTTTTTCTCCTTGACGTTAAAGTATA |
GAL10p-F | TCAATATAGCAATGAGCAGTTAAGCGTA |
GAL10p-R | TTTCAAAAATTCTTACTTTTTTTTTGGATG |
CYC1t-F | ATCCGCTCTAACCGAAAAGGAA |
CYC1t-R | CTTCGAGCGTCCCAAAACCTT |
ADH1t-F | CGAATTTCTTATGATTTATGATTTTTATTATTAA |
AHD1t-R | GAGCGACCTCATGCTATACCTGAG |
MET-F | TCGCGCGTTTCGGTGATGAC |
MET-R | AAACTTTGTTGAATGTTGAGCAAGTTAACATC |
LEU-F | TCGAGGAGAACTTCTAGTATATCCACATACCTAATA |
LEU-R | TCGACTACGTCGTAAGGCCGTTTC |
实施例2 各表达模块的构建
1、利用重叠PCR构建基因表达模块I~VI
(1)各表达模块的构建均采用重叠PCR技术,模块中各连接的片段通过PCR克隆获得,相邻片段的引物设计40~50 bp的重叠区域,并且使重叠区域碱基的退火温度在60℃以上。
(2)第一轮PCR扩增反应体系:KAPA Taq ReadyMix(KK1006,默克) 10 μL,上下游引物各0.5 μL,ddH2O 7 μL,模板1 μL,总反应体系为20 μL;其中引物序列见表2。
第一轮PCR扩增反应条件为:98℃ 1 min;98℃ 30 s,55-60℃ 30 s,72℃ 1-4min,15循环;72℃ 10 min。
(3)取第一轮PCR扩增反应液1 μL作为第二轮PCR扩增反应的模板,第二轮PCR扩增反应体系:KAPA Taq ReadyMix(KK1006,默克) 10 μL,上下游引物各0.5 μL,ddH2O 7 μl,模板1 μL,总反应体系为20 μL;其中引物序列见表2。
第二轮PCR扩增反应条件:98℃ 1 min;98℃ 30 s,50~60℃ 30 s,72℃ 1-4min,32个循环;72℃ 10 min。
(4)凝胶电泳胶回收目的片段,并连接pEASY-Blunt亚克隆载体(CB101,全式金生物技术有限公司)测序。
按上述步骤共构建以下六个模块:
(a)将GAL1p、tHMG1和ADH1t连接,构建基因表达模块GAL1p-tHMG1-ADH1t,命名为模块I。其中,第一轮PCR克隆PGK1p的引物为I-GtA-F1和I-GtA-R1,克隆tHMGR的引物为I-GtA-F2和I-GtA-R2,克隆ADH1t的引物为I-GtA-F3和I-GtA-R3;第二轮的PCR引物为I-GtA-F1和I-GtA-R3;引物序列见表2。
(b)将GAL10p、UPC2-1和CYC1t连接,构建基因表达模块GAL10p-UPC2-1-CYC1t,命名为模块II。其中,第一轮PCR克隆GAL10p的引物为II-GUC-F1和II-GUC-R1,克隆UPC2-1的引物为II-GUC-F2和II-GUC-R2,克隆CYC1t的引物为II-GUC-F3和II-GUC-R3;第二轮的PCR引物为II-GUC-F1和II-GUC-R3;引物序列见表2。
(c)将模块I和模块II连接,构建基因表达模块GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t,命名为模块III。其中,第一轮PCR克隆模块I的引物为III-GC-F1和III-GC-R1,克隆模块II的引物为III-GC-F2和III-GC-R2;第二轮的PCR引物为III-GC-F1和III-GC-R2;引物序列见表2。
(d)将MET筛选标记和HXT1p,构建基因表达模块MET-HXT1p,命名为模块IV;其中,第一轮PCR克隆MET筛选标记引物为IV-MH-F1和IV-MH-R1,克隆GAL1p引物为IV-MH-F2和IV-MH-R2;第二轮PCR引物为IV-MH-F1和IV-MH-R2;引物序列见表2。
(e)将LEU筛选标记和GAL10p,构建基因表达模块LEU-GAL10p,命名为模块V;其中,第一轮PCR克隆LEU筛选标记引物为V-LG-F1和V-LG-R1,克隆GAL10p引物为V-LG-F2和V-LG-R2;第二轮PCR引物为V-LG-F1和V-LG-R2;引物序列见表2。
(f)将GAL10p、ZSS2和CYC1t连接,构建基因表达模块GAL10p-ZSS2-CYC1t,命名为模块VI。其中,第一轮PCR克隆GAL10p的引物为VI-GZC-F1和VI-GZC-R1,克隆ZSS2的引物为VI-GZC-F2和VI-GZC-R2,克隆CYC1t的引物为VI-GZC-F3和VI-GZC-R3;第二轮的PCR引物为VI-GZC-F1和VI-GZC-R3;引物序列见表2。
2、酵母基因组整合型表达载体pCfB2875-III和pCfB2988-VI的构建
(1)利用限制性内切酶SfaAI和NheI酶切载体pCfB2875(Addgene),酶切体系如下:pCfB2875载体 1μL,限制性内切酶SfaAI(FD2094,默克) 0.5 μL,限制性内切酶NheI(FD0973,默克) 0.5 μL,10 x Fast Digest Buffer 1 μL,ddH2O 7 μL,总反应体系为10 μL。
(2)37℃酶切1 h,然后胶回收线性化的载体。
(3)将模块III从pEASY-Blunt-GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t(即上述模块III连接pEASY-Blunt载体获得)上扩增下来,胶回收获得纯化的PCR产物,然后将其同源重组到线性化的pCfB2875载体限制性内切酶酶切位点SfaAI和NheI之间,同源重组体系如下:Exnase® II 1 μL,5×CE II Buffer 2 μL,回收的线性化载体 1 μL,纯化的PCR产物 4 μL,ddH2O 2 μL,总反应体系为10 μL。
(4)37℃反应30 min,将同源重组体系转化DH5α感受态细胞,涂布平板,37℃培养过夜,经菌落PCR鉴定和质粒测序,得到整合型表达载体pCfB2875-III。
(5)带有模块VI的整合型表达载体pCfB2988-VI的构建方法同pCfB2875-III。
表2 实施例中构建各模块所用的引物序列
引物名称 | 引物序列(5’-3’) |
I-GtA-F1 | AGTACGGATTAGAAGCCGCCG |
I-GtA-R1 | TTCACCAATTGGTCTGCAGCCATGGGTTTTTTCTCCTTGACGTTAAAGTATA |
I-GtA-F2 | TATACTTTAACGTCAAGGAGAAAAAACCCATGGCTGCAGACCAATTGGTGAA |
I-GtA-R2 | TTAATAATAAAAATCATAAATCATAAGAAATTCGTTAGGATTTAATGCAGGTGACGGACC |
I-GtA-F3 | GGTCCGTCACCTGCATTAAATCCTAACGAATTTCTTATGATTTATGATTTTTATTATTAA |
I-GtA-R3 | GAGCGACCTCATGCTATACCTGAG |
II-GUC-F1 | TCAATATAGCAATGAGCAGTTAAGCGTA |
II-GUC-R1 | TGTGATTCTGTATACCGACTTCGCTCATTTTCAAAAATTCTTACTTTTTTTTTGGATG |
II-GUC-F2 | CATCCAAAAAAAAAGTAAGAATTTTTGAAAATGAGCGAAGTCGGTATACAGAATCACA |
II-GUC-R2 | TTCCTTTTCGGTTAGAGCGGATTCATAACGAAAAATCAGAGAAATTTGTTGTTG |
II-GUC-F3 | CAACAACAAATTTCTCTGATTTTTCGTTATGAATCCGCTCTAACCGAAAAGGAA |
II-GUC-R3 | CTTCGAGCGTCCCAAAACCTT |
III-GC-F1 | AGTACGGATTAGAAGCCGCCG |
III-GC-R1 | TACGCTTAACTGCTCATTGCTATATTGAGAGCGACCTCATGCTATACCTGAG |
III-GC-F2 | CTCAGGTATAGCATGAGGTCGCTCTCAATATAGCAATGAGCAGTTAAGCGTA |
III-GC-R2 | CTTCGAGCGTCCCAAAACCTT |
IV-MH-F1 | TCGCGCGTTTCGGTGATGAC |
IV-MH-R1 | CGGCGGCTTCTAATCCGTACTAAACTTTGTTGAATGTTGAGCAAGTTAACATC |
IV-MH-F2 | GATGTTAACTTGCTCAACATTCAACAAAGTTTAGTACGGATTAGAAGCCGCCG |
IV-MH-R2 | GGGTTTTTTCTCCTTGACGTTAAAGTATA |
V-LG-F1 | TCGAGGAGAACTTCTAGTATATCCACATACCTAATA |
V-LG-R1 | TACGCTTAACTGCTCATTGCTATATTGATCGACTACGTCGTAAGGCCGTTTC |
V-LG-F2 | GAAACGGCCTTACGACGTAGTCGATCAATATAGCAATGAGCAGTTAAGCGTA |
V-LG-R2 | TTTCAAAAATTCTTACTTTTTTTTTGGATG |
VI-GZC-F1 | TCAATATAGCAATGAGCAGTTAAGCGTA |
VI-GZC-R1 | TCGAAGGTCAAAGACTGCTTTTCCATTTTCAAAAATTCTTACTTTTTTTTTGGATG |
VI-GZC-F2 | CATCCAAAAAAAAAGTAAGAATTTTTGAAAATGGAAAAGCAGTCTTTGACCTTCGA |
VI-GZC-R2 | TTCCTTTTCGGTTAGAGCGGATTTACTTGTTAAAGTAATCGCAGGATTCAAC |
VI-GZC-F3 | GTTGAATCCTGCGATTACTTTAACAAGTAAATCCGCTCTAACCGAAAAGGAA |
VI-GZC-R3 | CTTCGAGCGTCCCAAAACCTT |
实施例3、构建工程酵母菌(产β-桉叶醇工程菌)
1、酿酒酵母菌株ZZ01的构建
(1)利用限制性内切酶NotI酶切构建好的整合型表达载体pCfB2875-III,凝胶电泳检测并回收目的条带,获得DNA整合片段S1。
(2)取50 μL上述纯化的DNA片段,转化酿酒酵母BY4741,以将模块整合到酵母BY4741染色体的TY3位点,并用尿嘧啶缺陷型培养基(SD-URA培养基)平板进行筛选,经菌落PCR验证,得到阳性工程菌株BY4741-1。
(3)将pSH65载体(武汉淼灵生物科技有限公司)转化BY4741-1菌株,用含有100 μg/mL博来霉素的YPD固体培养基进行筛选。
(4)将单克隆挑取到含有100 μg/mL博来霉素的YPD培养基中进行培养。
(5)培养四天后,采用划线稀释的方法,获得单克隆菌落。
(6)提取单克隆菌落的基因组,PCR扩增整合的DNA片段S1,经测序筛选得到整合型载体KIURA3筛选标记被敲除的菌株,再通过YPD培养基传代培养十天,丢掉pSH65质粒,得到工程菌株ZZ01。
2、酿酒酵母菌株ZZ02的构建
(1)将模块IV从pEASY-Blunt-MET-HXT1p上扩增下来,PCR反应体系:KAPA TaqReadyMix(KK1006,默克) 25 μL,上下游引物各1.25 μL,ddH2O 21.5 μl,模板1 μL,总反应体系为50 μL。
PCR扩增反应条件为:98℃ 1 min;98℃ 30 s,55-60℃ 30 s,72℃ 2 min,32循环;72℃ 10 min。
跑电泳检测PCR产物,并纯化回收。
(2)将回收的PCR片段转化菌株ZZ01,用甲硫氨酸(SD-MET培养基)平板进行筛选,得到酿酒酵母工程菌株ZZ02。
3、酿酒酵母菌株ZZ03的构建
(1)将模块V从pEASY-Blunt-LEU-GAL10p上扩增下来,PCR反应体系:KAPA TaqReadyMix(KK1006,默克) 25 μL,上下游引物各1.25 μL,ddH2O 21.5 μl,模板1 μL,总反应体系为50 μL。
PCR扩增反应条件为:98℃ 1 min;98℃ 30 s,55-60℃ 30 s,72℃ 2 min,32循环;72℃ 10 min。
跑电泳检测PCR产物,并纯化回收。
(2)将回收的PCR片段转化菌株ZZ02,用甲硫氨酸和亮氨酸(SD-MET-LEU培养基)平板进行筛选,得到酿酒酵母工程菌株ZZ03。
4、生产β-桉叶醇的酿酒酵母菌株ZZ04的构建
(1)利用限制性内切酶NotI酶切构建好的整合型表达载体pCfB2988-VI,凝胶电泳检测并回收目的条带,获得DNA整合片段S2。
(2)取50 μL上述纯化的DNA片段,转化酿酒酵母菌株ZZ03,以将模块整合到酵母染色体的TY1位点,用甲硫氨酸、亮氨酸和尿嘧啶(SD-MET-LEU-URA培养基)的平板进行筛选,经菌落PCR验证,得到生产β-桉叶醇的酿酒酵母工程菌株ZZ04。
实施例4 菌株ZZ04发酵生成目的化合物
1、菌株ZZ04的发酵培养
(1)将菌株ZZ04的单菌落接种到5 mL的SD-MET-LEU-URA培养基中,30℃条件下220rpm培养OD值至2~3。
(2)将上述菌液接种到15 mL的YPD培养基中,30℃条件下220 rpm培养OD值至2~3。
(3)将上述菌液分别接种到三个100 mL发酵培养基中,30℃条件下220 rpm培养至OD600值至8~10。
(4)将上述300 mL菌液全部接种到5 L的发酵罐中(内含3 L发酵培养基)进行发酵,发酵条件控制如下:温度30℃,转速300~1000 rpm,通气量3~20 L/min,溶氧值30%,用氨水维持pH为5.0。
(5)当溶氧值达到60%时,开始进行补料(加入补料培养基),使培养基中的半乳糖含量维持在5 g/L,发酵培养144 h,最终得到发酵液。
2、目的产物的萃取与检测
(1)取上述发酵液,加入等体积的乙酸乙酯,超声1 h,然后静置48 h。
(2)取有机层于干净的液相小瓶中,进行GC-MS检测。其中,所用的仪器优选为安捷伦气质联用仪7890B-5977B。检测方法如下:进样体积为1 μL,溶剂延时设置为12 min,载气为氦气,流速为1 mL/min。色谱柱为HP-5MS。色谱条件:50℃,3 min;20℃/min速率升温到70℃,1 min;15℃/min升温到300℃,3 min。
(3)如图2所示,GC-MS检测结果显示,ZZ04菌株经发酵144 h可以产生100 mg/L 的β-桉叶醇。
上述实施例中所涉及的培养基的配方如下:
(1)YPD培养基:蛋白胨20 g/L,酵母提取物 10 g/L,葡萄糖20 g/L(固体YPD培养基在配制时添加20 g/L的琼脂粉)。
(2)SD-URA培养基:YPD培养基 6.7 g/L,URA(尿嘧啶)缺陷氨基酸(100 X)10 mL/L,葡萄糖20 g/L(固体培养基配制时添加20 g/L的琼脂粉)。
(3)SD-URA-MET培养基:YPD 培养基6.7 g/L,URA(尿嘧啶)和MET(甲硫氨酸)缺陷氨基酸(100 X)10 mL/L,葡萄糖20 g/L(固体培养基配制时添加20 g/L的琼脂粉)。
(4)SD-URA-MET-LEU培养基:YPD培养基 6.7 g/L,URA(尿嘧啶)、MET(甲硫氨酸)和LEU(亮氨酸)缺陷氨基酸(100 X)10 mL/L,葡萄糖20 g/L(固体培养基配制时添加20 g/L的琼脂粉)。
(5)SD-URA-MET-LEU-HIS培养基:YPD培养基 6.7 g/L,URA(尿嘧啶)、MET(甲硫氨酸)、LEU(亮氨酸)和HIS(组氨酸)缺陷氨基酸(100 X)10 mL/L,葡萄糖20 g/L(固体培养基配制时添加20 g/L的琼脂粉)。
HIS/MET/LEU/URA四缺氨基酸母液(100 X):精氨酸0.12 g,天冬氨酸 0.6 g,谷氨酸0.6 g,赖氨酸0.18 g,苯丙氨酸0.3 g,丝氨酸2.25 g,苏氨酸1.2 g,色氨酸0.24 g,酪氨酸0.18 g,缬氨酸0.9 g,用蒸馏水定容到57 mL,根据需要可不加任意一种氨基酸配置成缺陷氨基酸母液(100 X)。上述原料均购买自Sigma-Aldrich。
甲硫氨酸(MET)0.12 g,尿嘧啶(URA)0.12 g,亮氨酸(LEU)0.36 g,组氨酸(HIS)0.12 g
(6)发酵培养基:磷酸二氢钾(KH2PO4) 8 g/L,硫酸铵((NH4)2SO4) 15 g/L,硫酸镁(MgSO4) 3 g/L,七水硫酸锌(ZnSO4•7H2O) 0.72 g/L,维生素溶液12 mL/L,微量金属盐溶液10 mL/L,葡萄 25 g/L;其中:
维生素溶液的成分为:肌醇25 g/L,盐酸硫胺素1 g/L,盐酸吡哆醇1 g/L,烟酸1g/L,泛酸钙1 g/L,维生素H 0.05 g/L和对氨基苯甲酸0.2 g/L。
微量金属盐溶液的成分为:乙二胺四乙酸(EDTA) 15 g/L,七水硫酸锌(ZnSO4•7H2O) 10.2 g/L,四水氯化锰(MnCl2•4H2O) 0.5 g/L,硫酸铜(CuSO4) 0.5 g/L,六水氯化钴(CoCl2•6H2O) 0.86 g/L,二水钼酸钠(Na2MoO4•2H2O) 0.56 g/L,二水氯化钙(CaCl2•2H2O)3.84 g/L和七水硫酸铁(FeSO4•7H2O) 5.12 g/L。
(7)补料培养基:葡萄糖 585 g/L,磷酸二氢钾(KH2PO4)9 g/L,硫酸钾(K2SO4) 3.5g/L,硫酸镁(MgSO4) 2.5 g/L,硫酸钠(Na2SO4) 0.28 g/L,维生素溶液 12 mL/L,以及微量金属盐溶液10 mL/L;其中:
维生素溶液:肌醇25 g/L,盐酸硫胺素1 g/L,盐酸吡哆醇1 g/L,烟酸1 g/L,泛酸钙1 g/L,维生素H 0.05 g/L和对氨基苯甲酸0.2 g/L。
微量金属盐溶液:乙二胺四乙酸(EDTA) 15 g/L,七水硫酸锌(ZnSO4•7H2O) 10.2g/L,四水氯化锰(MnCl2•4H2O) 0.5 g/L,硫酸铜(CuSO4) 0.5 g/L,六水氯化钴(CoCl2•6H2O)0.86 g/L,二水钼酸钠(Na2MoO4•2H2O) 0.56 g/L,二水氯化钙(CaCl2•2H2O) 3.84 g/L和七水硫酸铁(FeSO4•7H2O) 5.12 g/L。
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本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 滨州医学院
烟台毓璜顶医院
<120> 一种产β-桉叶醇工程菌及其构建方法、应用
<141> 2022-05-16
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1584
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggctgcag accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60
caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120
agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat 180
tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240
ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300
ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg 360
gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420
ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480
tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat 540
ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600
atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac aagaggccca 660
gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa 720
gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa 780
catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840
gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900
gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960
aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta agagtgtcgt cgcagaagct 1020
actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080
ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac 1140
gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200
aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260
tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca 1320
caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380
aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440
tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct 1500
gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560
tccgtcacct gcattaaatc ctaa 1584
<210> 2
<211> 2742
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tgatatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 3
<211> 2742
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60
aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120
cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180
tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240
acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300
gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360
cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420
tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480
gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540
gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600
ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660
ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720
caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780
ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840
attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900
caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960
caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020
caacagcagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080
gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140
actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200
atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260
tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320
actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380
gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440
aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500
acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560
tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620
ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680
cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740
gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800
accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860
tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920
gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980
ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040
agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100
agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160
aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220
acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280
gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340
gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400
ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460
cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520
gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580
gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640
gacgaataca gtggaggtgg tggtatgcat atgatgctag atttcctcgg tggcggatta 2700
ccatcgatga caacaacaaa tttctctgat ttttcgttat ga 2742
<210> 4
<211> 1665
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggaaaagc agtctttgac cttcgatggt gatgaagaag ctaagatcga taggaagtcc 60
tctaaatacc atccatctat ttggggtgac tacttcatcc aaaactcttc attgacccat 120
gccaaagaat ctacccaaag aatgatcaag agagtcgaag aattgaaggt ccaagtcaag 180
tctatgttca aggatacctc tgacttgttg cagttgatga acttgatcaa ctccatccaa 240
atgctaggtt tggattacca cttcgaaaac gaaattgatg aagccttgag gttgatctac 300
gaagttgatg ataagtctta cggcttgtac gaaacctctt tgagatttca gttgttgaga 360
caacatggct atcatgtttc cgccgatatt ttcaacaagt tcaaggacga taacggctcc 420
ttcatttctt ctttgaatgg tgatgctaag ggcctgttgt ccttgtataa tgtttcttac 480
ttgggtactc acggcgaaac tattttagat gaagctaagt ctttcacgaa gccccaattg 540
gtttctttga tgtctgaatt ggaacaatcc ttggctgctc aagtttcttt gtttttggaa 600
ttgccactgt gcaggcgtaa caagattttg ttggctagaa agtacatcct gatctaccaa 660
gaggatgcta tgagaaacaa cgtcatattg gaattggcca agctgaactt caacctgttg 720
caatcattat accaagaaga gttgaagaaa atctccatct ggtggaatga tttggctttt 780
gccaagtctt tgtctttcac cagagataga gttgtcgaag gttattactg ggttttgacc 840
atctacttcg aaccacaaca ttcaagagct agagttatct gctctaaggt tttcgccttc 900
ttgtccatta tggatgacat ctacgataac tacggtatct tggaagaatg taccttgttg 960
accgaagcta ttaagagatg gaatccacaa gctattgatg gtttgccaga atacttgaag 1020
gactactact tgaagttgct gaaaaccttc gaggaatttg aggatgaatt ggagttgaac 1080
gagaagtaca gaatgttgta cttgcaagat gaagttaagg ccttggctat ctcttactta 1140
caagaggcta aatggggtat cgaaagacat gttccatcat tggatgaaca cttgcacaac 1200
tcattgatct cttcaggttc ttctaccgtt atttgcgctt cttttgttgg tatgggtgaa 1260
gttgccacca aagaagtttt tgattggttg tcatctttcc caaaggttgt tgaagcctgt 1320
tgtgttatcg gtagactgtt gaacgatatt aggtcacatg aactggaaca aggtagagat 1380
catactgctt ctactgtcga atcttacatg aaggaacatg ataccaacgt tgatgttgct 1440
tgcgaaaagt tgagagaaat cgttgaaaag gcttggaagg acttgaacaa cgaatctttg 1500
aatccaacta aggtcccaag gttgatgatc gaaagaatcg ttaacctgtc caagtccaac 1560
gaagaaatct acaagtacaa tgacacctac accaactctg atacaaccat gaaggataac 1620
atctcattgg tcttggttga atcctgcgat tactttaaca agtaa 1665
<210> 5
<211> 1123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgcaggtctc atctggaata taattccccc ctcctgaagc aaatttttcc tttgagccgg 60
aatttttgat attccgagtt ctttttttcc attcgcggag gttattccat tcctaaacga 120
gtggccacaa tgaaacttca attcatatcg accgactatt tttctccgaa ccaaaaaaat 180
agcagggcga gattggagct gcggaaaaaa gaggaaaaaa ttttttcgta gttttcttgt 240
gcaaattagg gtgtaaggtt tctagggctt attggttcaa gcagaagaga caacaattgt 300
aggtcctaaa ttcaaggcgg atgtaaggag tattggtttc gaaagttttt ccgaagcggc 360
atggcaggga ctacttgcgc atgcgctcgg attatcttca tttttgcttg caaaaacgta 420
gaatcatggt aaattacatg aagaattctc tttttttttt tttttttttt ttttttacct 480
ctaaagagtg ttgaccaact gaaaaaaccc ttcttcaaga gagttaaact aagactaacc 540
atcataactt ccaaggaatt aatcgatatc ttgcactcct gatttttctt caaagagaca 600
gcgcaaagga ttatgacact gttgcattga gtcaaaagtt tttccgaagt gacccagtgc 660
tctttttttt tttccgtgaa ggactgacaa atatgcgcac aagatccaat acgtaatgga 720
aattcggaaa aactaggaag aaatgctgca gggcattgcc gtgccgatct tttgtctttc 780
agatatatga gaaaaagaat attcatcaag tgctgataga agaataccac tcatatgacg 840
tgggcagaag acagcaaacg taaacatgag ctgctgcgac atttgatggc ttttatccga 900
caagccagga aactccacca ttatctaatg tagcaaaata tttcttaaca cccgaagttg 960
cgtgtccccc tcacgttttt aatcatttga attagtatat tgaaattata tataaaggca 1020
acaatgtccc cataatcaat tccatctggg gtctcatgtt ctttccccac cttaaaatct 1080
ataaagatat cataatcgtc aactagttga tatacgtaaa atc 1123
<210> 6
<211> 455
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact ctcctccgtg 60
cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc 120
cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt 180
aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag gatgataatg 240
cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg atttttgatc 300
tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac tttcaacatt 360
ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa attgttaata 420
tacctctata ctttaacgtc aaggagaaaa aaccc 455
<210> 7
<211> 212
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tcaatatagc aatgagcagt taagcgtatt actgaaagtt ccaaagagaa ggttttttta 60
ggctaagata atggggctct ttacatttcc acaacatata agtaagatta gatatggata 120
tgtatatgga tatgtatatg gtggtaatgc catgtaatat gattattaaa cttctttgcg 180
tccatccaaa aaaaaagtaa gaatttttga aa 212
<210> 8
<211> 165
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat 60
acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt 120
tcctgtaggt caggttgctt tctcaggtat agcatgaggt cgctc 165
<210> 9
<211> 190
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60
ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120
gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180
acgctcgaag 190
<210> 10
<211> 2842
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca attatttttt 240
gctttttctc ttgaggtcac atgatcgcaa aatggcaaat ggcacgtgaa gctgtcgata 300
ttggggaact gtggtggttg gcaaatgact aattaagtta gtcaaggcgc catcctcatg 360
aaaactgtgt aacataataa ccgaagtgtc gaaaaggtgg caccttgtcc aattgaacac 420
gctcgatgaa aaaaataaga tatatataag gttaagtaaa gcgtctgtta gaaaggaagt 480
ttttcctttt tcttgctctc ttgtcttttc atctactatt tccttcgtgt aatacagggt 540
cgtcagatac atagatacaa ttctattacc cccatccata caatgccatc tcatttcgat 600
actgttcaac tacacgccgg ccaagagaac cctggtgaca atgctcacag atccagagct 660
gtaccaattt acgccaccac ttcttatgtt ttcgaaaact ctaagcatgg ttcgcaattg 720
tttggtctag aagttccagg ttacgtctat tcccgtttcc aaaacccaac cagtaatgtt 780
ttggaagaaa gaattgctgc tttagaaggt ggtgctgctg ctttggctgt ttcctccggt 840
caagccgctc aaacccttgc catccaaggt ttggcacaca ctggtgacaa catcgtttcc 900
acttcttact tatacggtgg tacttataac cagttcaaaa tctcgttcaa aagatttggt 960
atcgaggcta gatttgttga aggtgacaat ccagaagaat tcgaaaaggt ctttgatgaa 1020
agaaccaagg ctgtttattt ggaaaccatt ggtaatccaa agtacaatgt tccggatttt 1080
gaaaaaattg ttgcaattgc tcacaaacac ggtattccag ttgtcgttga caacacattt 1140
ggtgccggtg gttacttctg tcagccaatt aaatacggtg ctgatattgt aacacattct 1200
gctaccaaat ggattggtgg tcatggtact actatcggtg gtattattgt tgactctggt 1260
aagttcccat ggaaggacta cccagaaaag ttccctcaat tctctcaacc tgccgaagga 1320
tatcacggta ctatctacaa tgaagcctac ggtaacttgg catacatcgt tcatgttaga 1380
actgaactat taagagattt gggtccattg atgaacccat ttgcctcttt cttgctacta 1440
caaggtgttg aaacattatc tttgagagct gaaagacacg gtgaaaatgc attgaagtta 1500
gccaaatggt tagaacaatc cccatacgta tcttgggttt cataccctgg tttagcatct 1560
cattctcatc atgaaaatgc taagaagtat ctatctaacg gtttcggtgg tgtcttatct 1620
ttcggtgtaa aagacttacc aaatgccgac aaggaaactg acccattcaa actttctggt 1680
gctcaagttg ttgacaattt aaagcttgcc tctaacttgg ccaatgttgg tgatgccaag 1740
accttagtca ttgctccata cttcactacc cacaaacaat taaatgacaa agaaaagttg 1800
gcatctggtg ttaccaagga cttaattcgt gtctctgttg gtatcgaatt tattgatgac 1860
attattgcag acttccagca atcttttgaa actgttttcg ctggccaaaa accatgagtg 1920
tgcgtaatga gttgtaaaat tatgtataaa cctactttct ctcacaagta ctatactttt 1980
ataaaacgaa ctttattgaa atgaatatcc tttttttccc ttgttacatg tcgtgactcg 2040
tactttgaac ctaaattgtt ctaacatcaa agaacagtgt taattcgcag tcgagaagaa 2100
aaatatggtg aacaagactc atctacttca tgagactact ttacgcctcc tataaagctg 2160
tcacactgga taaatttatt gtaggaccaa gttacaaaag aggatgatgg aggtttcttt 2220
acaataaaga agcacatgtg tgttaacgtt tttagtattt gcttgttatg taaatcagga 2280
aaacttcgcg ggatttggtt ggatgctact ttccatacaa taaatattat agatctaaaa 2340
agccaaatta caagtaaaga ttagtaaagc tgttggaatt ccatcgttga taaaaatgtt 2400
agttattaaa tataaaagtc agaataggtg aacttggatt taattgttgg catttcgttg 2460
ctgctagagg ccataatatt agatagccag gacatactag ttctcctcgt ggtataggaa 2520
tccataaaat ggaattggtg attctatgtg atatattcac attcttacta cattatcaat 2580
ccttgcactt cagcttcctc taacctcgat gacatcttct cataacttat gtcatcatct 2640
aacgccgtct attataatat attgatagta taagtattag ttgatagaca atagtggatt 2700
tttattccaa cagtgtcttt gttcgtctca gatatagtcg gattgccctt ttaagcaatc 2760
aatagtgttt tatttgcaac aatgtcgtca tagtttaata tgtcctataa gatgttaact 2820
tgctcaacat tcaacaaagt tt 2842
<210> 11
<211> 2228
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tcgaggagaa cttctagtat atccacatac ctaatattat tgccttatta aaaatggaat 60
cccaacaatt acatcaaaat ccacattctc ttcaaaatca attgtcctgt acttccttgt 120
tcatgtgtgt tcaaaaacgt tatatttata ggataattat actctatttc tcaacaagta 180
attggttgtt tggccgagcg gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt 240
aactgtggga atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta 300
tttttttcct caacataacg agaacacaca ggggcgctat cgcacagaat caaattcgat 360
gactggaaat tttttgttaa tttcagaggt cgcctgacgc atataccttt ttcaactgaa 420
aaattgggag aaaaaggaaa ggtgagaggc cggaaccggc ttttcatata gaatagagaa 480
gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc 540
tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt 600
acatttcagc aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctatg 660
tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc 720
gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa gttcgatttc 780
gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgtcccact tccagatgag 840
gcgctggaag cctccaagaa ggttgatgcc gttttgttag gtgctgtggc tggtcctaaa 900
tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa 960
ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga cttatctcca 1020
atcaagccac aatttgctaa aggtactgac ttcgttgttg tcagagaatt agtgggaggt 1080
atttactttg gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga tagtgaacaa 1140
tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat 1200
gagccaccat tgcctatttg gtccttggat aaagctaatc ttttggcctc ttcaagatta 1260
tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa ggttcaacat 1320
caattgattg attctgccgc catgatccta gttaagaacc caacccacct aaatggtatt 1380
ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt tatcccaggt 1440
tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa caccgcattt 1500
ggtttgtacg aaccatgcca cggttctgct ccagatttgc caaagaataa ggttgaccct 1560
atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa 1620
ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggtatcag aactggtgat 1680
ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa 1740
atccttgctt aaaaagattc tcttttttta tgatatttgt acataaactt tataaatgaa 1800
attcataata gaaacgacac gaaattacaa aatggaatat gttcataggg tagacgaaac 1860
tatatacgca atctacatac atttatcaag aaggagaaaa aggaggatag taaaggaata 1920
caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt gcactttaac 1980
attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag aaaatcatga 2040
aactacgatt cctaatttga tattggagga ttttctctaa aaaaaaaaaa atacaacaaa 2100
taaaaaacac tcaatgacct gaccatttga tggagtttaa gtcaatacct tcttgaagca 2160
tttcccataa tggtgaaagt tccctcaaga attttactct gtcagaaacg gccttacgac 2220
gtagtcga 2228
Claims (6)
1.一种产β-桉叶醇工程菌,其特征在于,所述产β-桉叶醇工程菌以酿酒酵母为出发菌株,过表达tHMGR、UPC2-1、ASG1和ZSS2基因,敲低ERG9基因;其中,
tHMGR和UPC2-1基因整合到酿酒酵母染色体TY3位点;
ERG9基因的启动子替换为半乳糖抑制型启动子HXT1p;
ASG1基因的启动子替换为组成型强启动子GAL1p;
ZSS2基因整合到酿酒酵母染色体的TY1位点;
所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1;
所述tHMGR和UPC2基因的核苷酸序列分别如SEQ ID NO.1~2所示;
所述UPC2-1基因的核苷酸序列如SEQ ID NO.3所示;
所述ZSS2基因的核苷酸序列如SEQ ID NO.4所示;
所述启动子HXT1p和GAL1p的核苷酸序列如SEQ ID NO.5~6所示。
2.根据权利要求1所述的产β-桉叶醇工程菌,其特征在于,所述酿酒酵母为酿酒酵母BY4741。
3.一种产β-桉叶醇工程菌的构建方法,其特征在于,包括如下步骤:
将GAL1p、tHMGR和ADH1t连接,构建基因表达模块I;
将GAL10p、UPC2-1和CYC1t连接,构建基因表达模块II;所述UPC2-1基因是基于PCR的定点突变技术将转录因子基因UPC2突变为UPC2-1;
将所述基因表达模块I和基因表达模块II连接,构建基因表达模块III;
将筛选标记MET和启动子HXT1p连接,构建基因表达模块IV;
将筛选标记LEU和启动子GAL10p连接,构建基因表达模块V;
将GAL10p、ZSS2和CYC1t连接,构建基因表达模块VI;
将基因表达模块III插入到载体pCfB2875的SfaSI酶切位点,得到整合表达载体pCfB2875-III;
用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2875-III,得到DNA整合片段S1;
将所述DNA整合片段S1整合到酿酒酵母染色体的TY3位点,得到菌株ZZ01;
将基因表达模块IV转化所述菌株ZZ01,得到菌株ZZ02;
将基因表达模块V转化所述菌株ZZ02,得到菌株ZZ03;
将基因表达模块VI插入到载体pCfB2988的SfaSI酶切位点,得到整合表达载体pCfB2988-VI;
用限制性核酸内切酶Not Ι酶切整合表达载体pCfB2988-VI,得到DNA整合片段S2;
将所述DNA整合片段S2整合到酿酒酵母染色体的TY1位点,得到产β-桉叶醇工程菌;
所述的tHMGR和UPC2基因的核苷酸序列分别如SEQ ID NO.1~2所示;
所述UPC2-1基因的核苷酸序列如SEQ ID NO.3所示;
所述ZSS2基因的核苷酸序列如SEQ ID NO.4所示;
所述启动子HXT1p、GAL1p和GAL10p的核苷酸序列如SEQ ID NO.5~7所示;
所述ADH1t和CYC1t的核苷酸序列如SEQ ID NO.8~9所示;
所述筛选标记MET和LEU的核苷酸序列如SEQ ID NO.10-11所示。
4.根据权利要求3所述的产β-桉叶醇工程菌的构建方法,其特征在于,所述酿酒酵母为酿酒酵母BY4741。
5.一种根据权利要求1-2任一所述的产β-桉叶醇工程菌或者根据权利要求3-4任一所述的产β-桉叶醇工程菌的构建方法所构建得到的产β-桉叶醇工程菌在生产β-桉叶醇中的应用。
6.一种β-桉叶醇的生产方法,其特征在于,包括:
将权利要求1-2任一所述的产β-桉叶醇工程菌或者根据权利要求3-4任一所述的产β-桉叶醇工程菌的构建方法所构建得到的产β-桉叶醇工程菌经活化后接种到发酵培养基中进行发酵培养,得到β-桉叶醇。
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