CN114107074B - 一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用 - Google Patents
一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种过表达3‑酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用,该方法包括以下步骤:以Schizochytrium ATCC 1381基因组DNA为模板,SEQ ID NO:2和SEQ ID NO:3所示的序列为引物,PCR扩增出3‑酮酰基合酶基因;将3‑酮酰基合酶基因插入pBlue‑MAT‑Zeo质粒中,获得过表达载体pBlue‑KSb;将过表达载体pBlue‑KSb转化高糖驯化裂殖壶菌HG‑20中,获得过表达3‑酮酰基合酶基因的裂殖壶菌基因工程菌株。由此以裂殖壶菌高糖驯化菌株为受体菌株,通过过表达3‑酮酰基合酶基因以提高重组菌中二十碳五烯酸EPA的占比。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用。
背景技术
ω-3系列多不饱和脂肪酸(Polyunsaturated Fatty Acids,PUFAs)如DHA、EPA和ALA等是具有生理活性和营养功能的脂肪酸,其对于降低胆固醇,降低心血管疾病风险等具有积极意义。
裂殖壶菌是属于破囊壶菌科的一类海洋真菌,富含多不饱和脂肪酸,已经应用于工业上生产DHA。但其合成多不饱和脂肪酸的完整途径仍不清晰,尤其是合成EPA的途径,尚处于探究阶段。裂殖壶菌的脂肪酸合成途径主要有FAS(Fatty acid synthase,FAS)和PKS(Polyketide synthase,PKS)。FAS是以一系列特异性催化的去饱和酶和延长酶的需氧途径合成长链脂肪酸,而PKS是一种边延长边去饱和的厌氧途径生产多不饱和脂肪酸。
相对于脂肪酸合成过程和涉及的酶类研究较为透彻的FAS途径,PKS途径迄今发现的所有多不饱和脂肪酸合成酶都是由三到四个亚基组成的超大多功能酶复合物,裂殖壶菌中完整的PKS途径合成多不饱和脂肪酸机理尚不明确。
因此,实现裂殖壶菌EPA产能的提升,有待改进。
发明内容
本发明旨在至少在一定程度上解决上述技术中的技术问题之一,提供一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用。本发明以裂殖壶菌高糖驯化菌株(HG-20)为受体菌株,通过过表达3-酮酰基合酶基因(KS)以提高重组菌中二十碳五烯酸EPA的占比。
为此,在本发明的实施例中,本发明在一方面提出了一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法,其包括以下步骤:
(1)以Schizochytrium ATCC 1381基因组DNA为模板,SEQ ID NO:2和SEQ ID NO:3所示的序列为引物,PCR扩增出3-酮酰基合酶基因KSb;
(2)将3-酮酰基合酶基因KSb插入pBlue-MAT-Zeo质粒中,获得过表达载体pBlue-KSb;
(3)将所述过表达载体pBlue-KSb转化高糖驯化裂殖壶菌HG-20中,获得过表达3-酮酰基合酶基因KSb的裂殖壶菌基因工程菌株。
根据本发明的实施例的一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法,该方法将裂殖壶菌PKS油脂合成基因簇中B亚基上的3-酮酰基合酶(3-ketoacyl synthase,KS)基因KSb克隆构建重组表达载体,通过电转化的方法将目的基因线性化片段导入到裂殖壶菌高糖驯化菌株中并整合到裂殖壶菌基因组中,构建了工程菌HG-KSb。该工程菌在发酵过程中,C16:0占比在96h时较HG-20下降了30.85%,而EPA在120h时提升了220.10%。
在本发明的第二方面中,提供了一种提高裂殖壶菌中EPA占比的方法,其特征在于,将如权利要求1所述的过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法构建的裂殖壶菌基因工程菌株接种于种子培养基活化后,获得发酵用菌种;将所述发酵菌种接种于发酵培养基中进行发酵培养,培养过程中取样进行油脂组分鉴定分析。
根据本发明实施例的一种提高裂殖壶菌中EPA占比的方法,该方法利用上述工程菌HG-KSb可提高裂殖壶菌中EPA占比,例如在培养过程中,C16:0占比在96h时较HG-20下降了30.85%,而EPA在120h时提升了220.10%。
可选地,将所述裂殖壶菌基因工程菌株接种至pH为6.4~6.6的新鲜的种子培养基中,于温度27~29℃转速180~220rpm的条件下培养48~96h,获得一级种子;将所述一级种子以2~10%的接种量接种至pH为6.4~6.6的新鲜的种子培养基中,于温度28℃转速180~220rpm的条件下培养36~48h,获得二级种子;将所述二级种子以2~10%的接种量接种至pH为6.4~6.6且葡萄糖的含量为60g/L的新鲜的发酵培养基中,于温度27~29℃转速180~220rpm的条件下培养5d,培养过程中取样进行油脂组分鉴定分析。
可选地,所述种子培养基的配方包括:葡萄糖150g/L、酵母粉10g/L、硫酸钠12g/L、氯化钾0.5g/L、硫酸镁2g/L、硫酸钾0.65g/L、磷酸二氢钾1g/L、硫酸铵1g/L、二水合氯化钙0.17g/L。
可选地,所述发酵培养基的配方包括:葡萄糖58~62g/L、谷氨酸钠4.8~5.1g/L、固体玉米浆粉4.8~5.1g/L、硫酸钠11~13g/L、氯化钾0.45~0.55g/L、硫酸镁1.9~2.1g/L、硫酸钾0.60~0.70g/L、磷酸二氢钾0.8~1.1g/L、硫酸铵0.8~1.1g/L、二水合氯化钙0.16~0.18g/L、七水合硫酸锌2.9~3.1mg/L、六水合氯化钴0.035~0.042mg/L、五水合硫酸铜1.9~2.1mg/L、六水合硫酸镍1.9~2.1mg/L、七水合硫酸铁9.5~10.3mg/L、四水合氯化锰2.9~3.1mg/L、二水合钼酸钠0.035~0.042mg/L、泛酸钙3.15~3.25mg/L、维生素B19.4~9.6mg/L、维生素B12 0.14~0.16mg/L。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1为本发明实施例中过表达工程菌成功构建的凝胶电泳验证结果图,其中泳道1:工程菌HG-KSb基因组博来霉素表达框PCR结果;泳道2:HG-20基因组博来霉素表达框PCR结果;泳道3:载体质粒pBlue-KSb博来霉素表达框PCR结果。Marker:TAKARA DL5000DNAMarker;
图2为本发明实施例的裂殖壶菌高糖驯化菌株HG-20及其过表达KSb基因工程菌HG-KSb的EPA含量比较图;
图3为本发明实施例的裂殖壶菌高糖驯化菌株HG-20及其过表达KSb基因工程菌HG-KSb的C16:0含量比较图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
下文的公开提供了许多不同的实施例或例子用来实现本发明的不同实施方式。为了简化本发明的公开,下文中对特定实施例或示例进行描述。当然,他们仅仅为示例,并且目的不在于限制本发明。此外,本发明提供的各种特定工艺和材料的例子,本领域普通技术人员可以意识到其他工艺的可应用性和/或其他材料的使用。除非另有说明,本发明的实施将采用本领域技术人员的能力范围之内的化学、分子生物学等领域的传统技术。另外,除非另有说明,在本文中,核酸以5′至3′的方向从左向右书写,氨基酸序列则以氨基端到羧基端的方向从左向右书写。
需要说明的是,以下实施例中:
种子培养基的配方为:葡萄糖150g/L、酵母粉9~11g/L、硫酸钠11~13g/L、氯化钾0.4~0.6g/L、硫酸镁1.8~2.2g/L、硫酸钾0.60~0.70g/L、磷酸二氢钾0.8~1.2g/L、硫酸铵0.8~1.2g/L、二水合氯化钙0.16~0.18g/L。
发酵培养基的配方为:葡萄糖60g/L、谷氨酸钠5g/L、固体玉米浆粉5g/L、硫酸钠12g/L、氯化钾0.5g/L、硫酸镁2g/L、硫酸钾0.65g/L、磷酸二氢钾1g/L、硫酸铵1g/L、二水合氯化钙0.17g/L、七水合硫酸锌3mg/L、六水合氯化钴0.04mg/L、五水合硫酸铜2mg/L、六水合硫酸镍2mg/L、七水合硫酸铁10mg/L、泛酸钙3.2mg/L、四水合氯化锰3mg/L、二水合钼酸钠0.04mg/L、维生素B1 9.5mg/L、维生素B12 0.15mg/L。
裂殖壶菌高糖驯化菌株是以购自美国模式培养物集存库中的裂殖壶菌ATCC-1381进行驯化,其驯化的具体方法可参考CN 109913513 B中记载的方式。
下面通过说明性的具体实施例对本发明进行描述,这些实施例并不以任何方式限制本发明的范围。特别说明的是:本发明所用到的试剂除特别说明外均有市售。
实施例1重组表达载体pBlue-KSb的构建
设计PCR引物,用于扩增KSb基因片段。
引物1:GGGCCCTCTCAAAGATTAAGGCA(SEQ ID NO:2,下划线为ApaI酶切位点)
引物2:CCACTAGTGCGGTCAAACTCC(SEQ ID NO:3,下划线为SpeI酶切位点)
以Schizochytrium ATCC 1381基因组DNA为模板,进行如下PCR程序:(1)95℃,3min;(2)95℃,15s;(3)56~72℃,15s;(4)72℃,30~60s/kb;(2)-(4)步重复30~35个循环;(5)72℃,5~10min,4℃保存。
PCR反应体系如下表:
将PCR产物和表达载体pBlue-MAT-Zeo(Li Z.,et al.Overexpression ofmalonyl-CoA:ACP transacylase in Schizochytrium sp.to improve polyunsaturatedfatty acids production[J].Journal of Agricultural and Food Chemistry,2018,66(21):5382-5391.)分别用限制性内切酶ApaI和SpeI双酶切,回收纯化后,将PCR产物和表达载体以3:1的比例用T4 DNA连接酶在4℃连接12h,构建重组表达载体pBlue-KSb(负载了核酸序列如SEQ ID NO:1所示的3-酮酰基合成酶KSb基因)。
实施例2过表达KSb基因的裂殖壶菌高糖驯化工程菌的构建
将重组表达载体pBlue-KSb经提取,并用ApaI和NotI双酶切线性化回收后,电击转入裂殖壶菌,28℃复苏3~4h后,涂布博来霉素(Zeocin)抗性平板,再在28℃培养3-5d,筛选转化子。转化子经基因组提取后,利用PCR进行验证(如图1所示)。从而获得过表达3-酮酰基合成酶KSb基因的裂殖壶菌高糖驯化工程菌HG-KSb。
电转化具体步骤如下:
裂殖壶菌感受态的制备:
(1)在20mL种子培养基中接种存在甘油管中的裂殖壶菌HG-20(高糖驯化菌株),接种量为2~10%,于28℃的温度及200rpm的转速下,培养48h,获得一级种子;
(2)取上述一级种子1mL接入20mL种子培养基中,于28℃的温度及200rpm的转速下,培养至对数生长期,直到细胞OD600达到0.5~0.8;
(3)取10mL菌液于已灭菌的50mL离心管中,3600g,4℃离心5min,弃上清;
(4)加入20mL预冷的无菌水,重悬,3600g,4℃离心5min弃上清;
(5)加入20mL预冷的含12.5mM DTT的0.2M磷酸氢二钠-磷酸二氢钠(v:v≈1:2)缓冲液(pH 6.5),28℃,200rpm震荡30min;
(6)振荡完毕后,3600g,4℃离心5min,弃上清;
(7)加入20mL预冷的0.2M磷酸氢二钠-磷酸二氢钠(v:v≈1:2)缓冲液(pH 6.5)重悬,3600g,4℃离心5min,弃上清;
(8)加入1mL 1M山梨醇,移液枪吹打转移至1.5mL无菌离心管,4000rpm,4℃离心5min,弃上清;
(9)加入600-800μL 1M山梨醇,重悬,分装100μL至1.5mL无菌离心管,置于冰上备用。
裂殖壶菌的电转:
(1)将100μL感受态细胞和1~2μg pBlue-KSb线性化片段转移到冰上预冷的0.2cm间隙的电击池中,静置30min;
(2)以1500V、5.5~5.8ms电击脉冲转化;
(3)迅速往电击池中加入1mL含有1M山梨醇的种子培养基复苏,将菌悬液回收到2mL离心管中,于28℃,200rpm培养3~4h;
(4)将菌液以4000rpm,1min离心后弃去部分上清液,浓缩重悬后涂布于博来霉素抗性平板上,于28℃避光培养直到平板上出现菌落,获得过表达KSb的裂殖壶菌高糖驯化工程菌HG-KSb。
实施例3利用裂殖壶菌高糖驯化菌株及其基因工程菌发酵制备EPA
(1)将保存在甘油管中的裂殖壶菌HG-20和实施例2所得的裂殖壶菌高糖驯化基因工程菌筛选所得转化子HG-KSb接入20mL种子培养基中,接种量为2~10%,于28℃及200rpm的转速下,培养48h,获得一级种子;
(2)将上述一级种子接入20mL种子培养基中,接种量为5%,于28℃及200rpm的转速下,培养48h,获得二级种子;
(3)将上述二级种子接入50mL发酵培养基中,接种量为2~10%,于28℃及200rpm的转速下,培养5~7d,得发酵液;
(5)分别取2~5d发酵液测不同高糖浓度下总油脂含量,并对脂肪酸进行分析鉴定。
具体结果如图2、图3所示,工程菌HG-KSb在发酵过程中,C16:0占比在96h时较HG-20下降了30.85%,而EPA在120h时提升了220.10%。由此,通过裂殖壶菌PKS油脂合成基因簇中B亚基上的3-酮酰基合酶(3-ketoacyl synthase,KS)基因过表达,可提高裂殖壶菌中EPA的占比。
总油脂含量的测定方法如下:
(1)选取发酵液样品3mL于15mL离心管中,混匀后再加入4mL质量分数38%的浓盐酸。于60~70℃水浴加热40~50min至菌体消化完全。
(2)然后再加入3~5mL正己烷,震荡,再于20℃,3000rpm离心1min后,取上层分层部分的正己烷置于已称重的50mL离心管中。用正己烷重复震荡萃取,3000rpm离心1min取上层有机相,此过程重复3~5次,直至上层有机相无色。
(3)常温下N2吹干正己烷,称重,得到净油脂产量。
(4)提取得到油脂进行甲酯化,干燥,滤膜过滤,所得样品即可进行气相色谱分析。
分析鉴定的具体方法如下:
向提取得到油脂中添加5mL的0.5M的KOH-CH3OH溶液于65℃水浴至油滴溶解(大约10min),然后加入5mL 30%的三氟化硼乙醚,65℃水浴加热反应30min;冷却后加入5mL正己烷充分振荡,加40μL内标物(如十六烷乙酸),然后加入足量的饱和食盐水,静置待两液面分层后取上层正己烷相并用过量无水硫酸钠脱水,0.22μL孔径有机系滤膜过滤,所得样品即可进行气相色谱分析。
气相色谱检测条件如下:安捷伦气相色谱分析仪,使用SP2560盘;使用N2作为载体,初始柱温140℃,检测器温度250℃,柱箱温度250℃;升温程序:初始温度140℃保持2min,之后以每分钟上升10℃的速度提升温度到230℃,保持5min。
综上,根据本发明的实施例,通过对裂殖壶菌PKS油脂合成基因簇中B亚基上的3-酮酰基合酶(3-ketoacyl synthase,KS)基因过表达,可使工程菌HG-KSb在发酵过程中,C16:0占比在96h时较原始菌HG-20下降了30.85%,而EPA在120h时提升了220.10%,从而可提高裂殖壶菌中EPA的占比。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外,本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 厦门大学
<120> 一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用
<130> 无
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1335
<212> DNA
<213> 人工序列
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atggcctctc gcaagaatgt gagcgctgct cacgaaatgc acgacgagaa gcgcattgcc 60
gtggtgggca tggccgtgca atacgcgggc tgcaaagaca aggaagagtt ctggaaagta 120
gtcatgggcg gtgaggctgc atggactaag attagcgata aacgcctcgg atccaacaag 180
cgagccgagc acttcaaagc agagcgtagc aaatttgcag ataccttttg caacgagaac 240
tacggctgcg tcgatgactc cgtcgataac gaacacgagc ttctccttaa gctctccaag 300
aaggctctct ccgagacatc ggtctccgac tctacaaggt gcggtattgt gagcggatgc 360
ctgtcctttc ccatggacaa cctccagggc gaactcctca atgtgtacca aaaccacgtc 420
gaaaagaaac tcggcgctcg cgtcttcaag gatgcctcca agtggtccga gcgtgagcag 480
tcgcagaacc ccgaggctgg tgaccgccgc atctttatgg acccggcatc cttcgtagca 540
gaagagctca acctcggtcc tcttcactac tctgtcgatg ctgcctgtgc caccgccctt 600
tacgtccttc gcctcgccca ggaccacctc gtttccggtg ctgctgatgt catgctcgct 660
ggtgcaactt gcttcccgga gccctttttc attctctccg gattctccac tttccaggcc 720
atgcctgtat cgggagacgg catctcgtac ccgcttcaca aggacagtca gggtctcacc 780
cctggtgaag gtggtgccat tatggttctc aagcgccttg acgacgctat tcgcgatgga 840
gaccacattt acggtactct gctcggtgct accatcagca atgctggctg tggtcttccc 900
ctcaagccgc acttgcccag cgagaagtcc tgcctcattg atacctacaa gcgcgtcaac 960
gtgcacccgc acaagatcca gtacgtcgag tgccacgcaa cgggtactcc ccagggagac 1020
cgcgttgaga ttgatgccgt caaggcttgc ttcgagggca aggtgcctcg ctttggaagc 1080
tccaagggta actttggcca cacactcgtt gcagctggtt tcgcaggcat gtgcaaggta 1140
ctccttgcca tgaagcatgg tgtgatcccg cccactcctg gtgtcgatgg atcttcccaa 1200
atggacccgc ttgtggtctc tgagcccatc ccatggcccg acactgaggg cgagcccaag 1260
cgcgctggtc tctccgcttt cggctttggt ggcaccaacg cccacgcagt ctttgaggag 1320
tttgaccgca ctagt 1335
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<400> 2
gggccctctc aaagattaag gca 23
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<400> 3
ccactagtgc ggtcaaactc c 21
Claims (3)
1.一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法,其特征在于,包括以下步骤:
(1)以裂殖壶菌 ATCC 1381基因组DNA为模板,SEQ ID NO:2和SEQ ID NO:3所示的序列为引物,PCR扩增出3-酮酰基合酶基因KSb,3-酮酰基合酶基因KSb的核酸序列如SEQ ID NO:1所示;
(2)将 3-酮酰基合酶基因KSb插入pBlue-MAT-Zeo质粒中,获得过表达载体pBlue-KSb;
(3)将所述过表达载体pBlue-KSb转化高糖驯化裂殖壶菌HG-20中,获得过表达3-酮酰基合酶基因KSb的裂殖壶菌基因工程菌株。
2.一种提高裂殖壶菌中EPA占比的方法,其特征在于,将如权利要求1所述的过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法构建的裂殖壶菌基因工程菌株接种于种子培养基活化后,获得发酵用菌种;将所述发酵菌种接种于发酵培养基中进行发酵培养,培养过程中取样进行油脂组分鉴定分析;所述种子培养基的配方为:葡萄糖150 g/L、酵母粉10 g/L、硫酸钠12 g/L、氯化钾0.5 g/L、硫酸镁2 g/L、硫酸钾0.65 g/L、磷酸二氢钾1g/L、硫酸铵1 g/L、二水合氯化钙0.17 g/L;所述发酵培养基的配方为:葡萄糖58~62 g/L、谷氨酸钠4.8~5.1 g/L、固体玉米浆粉4.8~5.1 g/L、硫酸钠11~13 g/L、氯化钾0.45~0.55g/L、硫酸镁1.9~2.1 g/L、硫酸钾0.60~0.70 g/L、磷酸二氢钾0.8~1.1 g/L、硫酸铵0.8~1.1g/L、二水合氯化钙0.16~0.18 g/L、七水合硫酸锌2.9~3.1 mg/L、六水合氯化钴0.035~0.042 mg/L、五水合硫酸铜1.9~2.1 mg/L、六水合硫酸镍1.9~2.1 mg/L、七水合硫酸铁9.5~10.3 mg/L、四水合氯化锰2.9~3.1 mg/L、二水合钼酸钠0.035~0.042 mg/L、泛酸钙3.15~3.25 mg/L、维生素B1 9.4~9.6 mg/L、维生素B12 0.14~0.16 mg/L。
3.如权利要求2所述的提高裂殖壶菌中EPA占比的方法,其特征在于,将所述裂殖壶菌基因工程菌株接种至pH为6.4~6.6的新鲜的种子培养基中,于温度27~29℃ 转速180~220rpm的条件下培养48~96 h,获得一级种子;将所述一级种子以2~10%的接种量接种至pH为6.4~6.6的新鲜的种子培养基中,于温度28℃ 转速180~220 rpm的条件下培养36~48 h,获得二级种子;将所述二级种子以2~10%的接种量接种至pH为6.4~6.6且葡萄糖的含量为60g/L的新鲜的发酵培养基中,于温度27~29℃ 转速180~220 rpm的条件下培养5 d,培养过程中取样进行油脂组分鉴定分析。
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