CN111454854A - 一株产虾青素的红冬孢酵母基因工程菌株 - Google Patents
一株产虾青素的红冬孢酵母基因工程菌株 Download PDFInfo
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- CN111454854A CN111454854A CN202010368747.8A CN202010368747A CN111454854A CN 111454854 A CN111454854 A CN 111454854A CN 202010368747 A CN202010368747 A CN 202010368747A CN 111454854 A CN111454854 A CN 111454854A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一株产虾青素的红冬孢酵母基因工程菌株,该菌株含有核苷酸序列如SEQ ID NO:1所示的β‑胡萝卜素羟化酶基因crtZ、核苷酸序列如SEQ ID NO:3所示的β‑胡萝卜素酮化酶基因crtW;本发明通过农杆菌介导法将β‑胡萝卜素酮化酶基因crtW和β‑胡萝卜素羟化酶基因crtZ转化到红冬孢酵母YM25235中构建红冬孢酵母基因工程菌株YM25235/pRHcrtW‑crtZ;该菌株表达的crtZ与crtW基因可将YM25235菌株中的β‑胡萝卜素进一步转化成虾青素,经发酵培养并与出发菌株相比,该工程菌株的虾青素产量可达到0.637mg/g干菌体,为大规模商业化生产虾青素奠定基础。
Description
技术领域
本发明属于生物技术领域,具体涉及一株产虾青素的红冬孢酵母基因工程菌株,本发明通过农杆菌介导法将β-胡萝卜素酮化酶基因crtW和β-胡萝卜素羟化酶基因crtZ转化到红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中构建红冬孢酵母基因工程菌株,使其进行功能表达,两个基因编码的蛋白酶可将YM25235菌株中的β-胡萝卜素转化成虾青素。
背景技术
虾青素 (化学名称:3,3′-二羟基-4,4′-二酮基-β-胡萝卜素),分子式为C40H52O4,分子量为596. 86;是一种在自然界中广泛存在的酮式类胡萝卜素。虾青素是类胡萝卜素合成的最高级别产物,在自然界虾青素具有很强的抗氧化活性,可以有效清除细胞内的氧自由基,同时还具有抗癌功能、显著的着色能力以及增强免疫力等功能。
基于以上的特性,虾青素在医药、水产养殖、保健及化妆品领域具有广泛的应用。目前市场上的虾青素来源主要为化学合成,不仅价格昂贵,而且同天然虾青素在结构、功能、应用及安全性等方面差别显著,因此对天然来源的虾青素的需求正在不断增加。
因此通过生物合成法生产虾青素是目前最具发展潜力的一种提取方法。微生物发酵生产虾青素只需要低成本天然底物作为碳源,该方法具有生物培养周期短、产量高、天然无污染等特点。利用微生物发酵生产的虾青素为反式结构,破壁后可直接作为饲料添加剂,且不存在由于季节性和地域性变化而导致的产量和市场问题;目前能够发酵产生虾青素的微生物主要有真菌、细菌和酵母菌等。因此,用微生物发酵法来替代其他方法可以从一定程度上缓解天然虾青素的生产发展局限,该方法将有助于实现发酵生产虾青素产品的工业化。
发明内容
针对虾青素生产中存在的问题,本发明提供了一株产虾青素的红冬孢酵母基因工程菌株,该菌株含有核苷酸序列如SEQ ID NO:1所示的β-胡萝卜素羟化酶基因crtZ、核苷酸序列如SEQ ID NO:3所示的β-胡萝卜素酮化酶基因crtW;
本发明β-胡萝卜素酮化酶(β-carotene ketolase)基因crtW和β-胡萝卜素羟化酶(β-carotene hydroxylase)基因crtZ来源于海生细菌副球菌(Paracoccus sp.)N81106菌株;β-胡萝卜素羟化酶(β-carotene hydroxylase)基因crtZ核苷酸序列如SEQ ID NO:1所示、或为该核苷酸序列的片段、或与SEQ ID NO:1互补的核苷酸序列,该基因序列长为489bp,该基因编码的氨基酸序列如SEQ ID NO:2所示的多肽或其片段;β-胡萝卜素酮化酶(β-carotene ketolase)基因crtW核苷酸序列如SEQ ID NO:3所示、或为该核苷酸序列的片段、或与SEQ ID NO:3互补的核苷酸序列,该基因序列长为729bp,该基因编码的氨基酸序列如SEQ ID NO:4所示的多肽或其片段。
重组表达载体的获取,是将SEQ ID NO:1与SEQ ID NO:3所示基因与质粒pRH2034连接构建重组载体;也可用本领域的技术人员熟知的方法来构建含β-胡萝卜素羟化酶基因crtZ和β-胡萝卜素酮化酶基因crtW的核苷酸序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等;所述β-胡萝卜素羟化酶基因crtZ和β-胡萝卜素酮化酶基因crtW的核苷酸序列可有效连接到表达载体的恰当启动子上,以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。
本发明利用化学合成法获得β-胡萝卜素羟化酶(β-carotene hydroxylase)基因crtZ和β-胡萝卜素酮化酶(β-carotene ketolase)基因crtW,通过无缝克隆的方法,将β-胡萝卜素羟化酶基因crtZ片段和β-胡萝卜素酮化酶基因crtW片段插入到 pRH2034质粒上,获得表达β-胡萝卜素羟化酶(β-carotene hydroxylase)基因crtZ和β-胡萝卜素酮化酶(β-carotene ketolase)基因crtW的质粒载体pRHcrtW-crtZ;然后将重组质粒pRHcrtW-crtZ转化至红冬孢酵母中,采用HPLC分析红冬孢酵母基因工程菌株的虾青素合成情况。
本发明的优点和技术效果:
红冬孢酵母YM25235菌株属于红冬孢酵母属(Rhodosporidium),该属的菌株都是产油红酵母,红冬孢酵母YM25235菌株自身不产虾青素,本发明通过代谢工程手段将β-胡萝卜素羟化酶(β-carotene hydroxylase)基因crtZ和β-胡萝卜素酮化酶(β-carotene ketolase)基因crtW导入YM25235菌株中进行功能表达,可进一步将β-类胡萝卜素转化成虾青素,有助于获得高产虾青素的红冬孢酵母基因工程菌株。目前这方面的研究和应用还未见报道。此外,红冬孢酵母还能快速高密度生长,可大量利用多种可再生底物和廉价原料进行发酵生产类胡萝卜素,经济学上具有很大优势,而且它们能在低pH条件下生长良好,有助于工业化应用过程中的细菌污染控制。
附图说明
图1为crtW基因的PCR扩增图;其中:1、DNA 分子量标记DL2000;2、为扩增产物;
图2为重组质粒pRHcrtW-crtZ限制性酶切分析;其中:1、DNA 分子量标记DL10000; 2、质粒pRH2034的NcoI和EcoRV双酶切;3、重组质粒pRHcrtW-crtZ的NcoI和EcoRV双酶切;4、crtW的PCR 产物;5、DNA 分子量标记DL2000;
图3为crtZ基因表达框Pgpd+crtZ+TtrpC的PCR扩增图;其中:1、DNA 分子量标记DL5000;2、为扩增产物;3、DNA 分子量标记DL2000;
图4为重组质粒pRHcrtW-crtZ限制性酶切分析;其中:1、DNA 分子量标记DL10000; 2、质粒pRH2034的KpnⅠ、Hind Ⅲ双酶切;3、重组质粒pRHcrtW-crtZ的KpnⅠ、Hind Ⅲ双酶切;4、crtZ基因表达框的PCR 产物;5、DNA 分子量标记DL2000;
图5为重组质粒pRHcrtW-crtZ的质粒图谱;
图6 重组质粒pRHcrtW-crtZ转化红冬孢酵母YM25235阳性克隆验证;1、DNA分子量标记DL2000;2、阴性对照;3、crtW基因PCR 产物;4、以重组质粒pRHcrtW-crtZ为模板的crtW基因PCR 产物;5、crtZ基因PCR 产物;6、以重组质粒pRHcrtW-crtZ为模板的crtZ基因PCR 产物;
图7为HPLC检测YM25235/pRHcrtW-crtZ菌株虾青素的生成;
图8为菌株YM25235/pRHcrtW-crtZ与对照菌株YM25235/pRH2034的各组分胡萝卜素含量。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法。
实施例1:利用无缝克隆的方法进行重组质粒pRHcrtW-crtZ的构建
使用Vazyme公司的ClonExpress® II One Step Cloning Kit试剂盒进行重组质粒pRHcrtW-crtZ的构建;根据说明书分别设计用于无缝克隆crtW、crtZ基因的特异性引物,以硕擎生物科技有限公司合成的目的片段为模板在PCR仪(BIOER公司)上进行PCR扩增,反应所用引物、组份和扩增条件如下:
pRHcrtW-F:5’- ACAACACCAGATCACTCACCATGGATGAGCGCACATGCC -3’,下划线为NcoI酶切位点;
pRHcrtW-R:5’- ATCCCGGTCGGCATCTACGATATCTCATGCGGTGTCCCC -3’ ,下划线为EcoRV酶切位点;
pRHcrtZ-F:5’- ATACATTATACGAACGGTACCTGTACAGTGACCGGT -3’,下划线为KpnⅠ酶切位点;
pRHcrtZ-R:5’- CTGATCCAAGCTCAAGCTAAGCTTGCATGCCTGCAG -3’,下划线为Hind Ⅲ酶切位点;
PCR扩增体系如下(50 μL):
扩增条件:94℃预变性5 min,再用94℃变性30 s,58℃退火30 s,72℃延伸130 s,进行30个循环,最后72℃彻底延伸10min,反应完后取产物1μL,然后在浓度为1%的琼脂糖凝胶中进行电泳分析,crtW基因扩增结果如图1所示,扩增得到大小约750bp的片段,crtZ基因扩增结果如图3所示,扩增得到大小约2200bp的片段,用琼脂糖凝胶DNA回收试剂盒(北京索莱宝科技有限公司)回收。在插入crtZ基因时,将表达载体pRH2034经KpnⅠ、Hind Ⅲ两个限制性内切酶进行酶切;在插入crtW基因时,将表达载体pRH2034经NcoI、EcoRV两个限制性内切酶进行酶切;随后将线性化表达载体与目的片段按一定比例混合后加入试剂盒提供的重组反应体系中即可获得重组质粒pRHcrtW-crtZ(图5)。将获得的重组质粒转入大肠杆菌 DH5α中扩增,再经菌落 PCR 验证后提取重组质粒,并用NcoI、EcoRV对pRHcrtW-crtZ进行双酶切验证;结果表明,重组质粒pRHcrtW-crtZ经双酶切后产生了0.75kb 和12 kb左右的两个条带(图 2 第3泳道),用Kpn Ⅰ、Hind Ⅲ对pRHcrtW-crtZ进行双酶切验证;结果表明,重组质粒pRHcrtW-crtZ经双酶切后产生了2.2kb 和10.6 kb左右的两个条带(图 4 第3泳道),与实验设计相符,初步表明重组质粒pRHcrtW-crtZ构建成功;用测序引物进行测序,将酶切验证正确的质粒送出测序进一步进行验证;测序结果表明,测序所获得的序列与目的序列完全一致,没有出现任何碱基突变和缺失等。
实施例2:红冬孢酵母基因工程菌株的构建以及红冬孢酵母中虾青素与类胡萝卜素的合成
1、农杆菌介导转化红冬孢酵母YM25235
利用农杆菌介导法将重组质粒pRHcrtW-crtZ转化至红冬孢酵母YM25235中,以含潮霉素B(Hygromycin B)终浓度为150µg/mL的 YPD 培养基筛选转化子,然后按照上海生工生物工程股份有限公司DNA 提取试剂盒说明书中步骤提取酵母转化子的基因组 DNA,后进行PCR验证,结果如图6所示;
2、利用HPLC进行重组质粒pRHcrtW-crtZ转化后红冬孢酵母中虾青素与类胡萝卜素合成含量的分析
将重组质粒pRHcrtW-crtZ转化后的菌株在28℃条件下培养144h,提取色素,并以转入空质粒pRH2034的红冬孢酵母菌株为对照,利用紫外-可见分光光度计在445nm下测定总类胡萝卜素的含量(mg/g干菌体);其中基因工程菌株YM25235/pRHcrtW-crtZ的总类胡萝卜素合成量比含空质粒pRH2034的对照菌株有明显提高,含空质粒pRH2034的对照菌株的类胡萝卜素合成量为5.503mg/g干菌体,而基因工程菌株YM25235/pRHcrtW-crtZ类胡萝卜素合成量为9.319mg/g干菌体,即基因工程菌株YM25235/pRHcrtW-crtZ类胡萝卜素合成量是对照菌的1.69倍。
使用HPLC,根据不同组分及标准品保留时间与光谱吸收值相结合的方法对色素产物进行定性与定量分析: 色素进样前需用0.45μm的微孔滤膜去除杂质;色谱柱:4.6×250mm 5μm ZORBAX Eclipse XDB-C18;流动相:A: 90%乙腈,10%水;B:100%乙酸乙酯;梯度洗脱:0~5min,B从0升至50%;5.01 min,B升至53%;5.01~10 min,B保持在53%;10~15.01 min,B升至54%;15.01~20 min,B升至55%;20.01 min,B升至56%;20.01~30 min,B从56%升至57%;30~35min,B降为0;流速1mL/min;进样量20μL;检测波长为445nm和480nm; 柱温:25℃;检测器:紫外多波长(VWD)检测器;经HPLC检测结果(图7)可知,YM25235/pRHcrtW-crtZ菌株的色素产物,在与虾青素标准品的同一保留时间下,可检测到虾青素的生成,虾青素产量可达0.637mg/ g干菌体,图8为菌株YM25235/pRHcrtW-crtZ与对照菌株YM25235/pRH2034的各组分胡萝卜素含量结果,从图中也可以看出在红冬孢酵母YM25235中表达β-胡萝卜素酮化酶基因crtW和β-胡萝卜素羟化酶基因crtZ能将YM25235菌株中的β-胡萝卜素进一步转化成虾青素;
结果表明,本发明红冬孢酵母基因工程菌株们能生产虾青素,目前也未见β-胡萝卜素酮化酶基因crtW和β-胡萝卜素羟化酶基因crtZ在红冬孢酵母YM25235中表达的报道。
序列表
<110> 昆明理工大学
<120> 一株产虾青素的红冬孢酵母基因工程菌株
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 489
<212> DNA
<213> 海生细菌副球菌N81106(Paracoccus sp. N81106)
<400> 1
atgaccaatt tcctgatcgt cgtcgccacc gtgctggtga tggagttgac ggcctattcc 60
gtccaccgct ggatcatgca cggccccctg ggctggggct ggcacaagtc ccaccacgag 120
gaacacgacc acgcgctgga aaagaacgac ctgtacggcc tggtctttgc ggtgatcgcc 180
acggtgctgt tcacggtggg ctggatctgg gcgccggtcc tgtggtggat cgccttgggc 240
atgactgtct atgggctgat ctatttcgtc ctgcatgacg ggctggtgca tcagcgctgg 300
ccgttccgtt atatcccgcg caagggctat gccagacgcc tgtatcaggc ccaccgcctg 360
caccatgcgg tcgaggggcg cgaccattgc gtcagcttcg gcttcatcta tgcgcccccg 420
gtcgacaagc tgaagcagga cctgaagatg tcgggcgtgc tgcgggccga ggcgcaggag 480
cgcacgtga 489
<210> 2
<211> 162
<212> PRT
<213> 海生细菌副球菌N81106(Paracoccus sp. N81106)
<400> 2
Met Thr Asn Phe Leu Ile Val Val Ala Thr Val Leu Val Met Glu Leu
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Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro Leu Gly Trp
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Gly Trp His Lys Ser His His Glu Glu His Asp His Ala Leu Glu Lys
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Asn Asp Leu Tyr Gly Leu Val Phe Ala Val Ile Ala Thr Val Leu Phe
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Thr Val Gly Trp Ile Trp Ala Pro Val Leu Trp Trp Ile Ala Leu Gly
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Met Thr Val Tyr Gly Leu Ile Tyr Phe Val Leu His Asp Gly Leu Val
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His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Lys Gly Tyr Ala Arg
100 105 110
Arg Leu Tyr Gln Ala His Arg Leu His His Ala Val Glu Gly Arg Asp
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His Cys Val Ser Phe Gly Phe Ile Tyr Ala Pro Pro Val Asp Lys Leu
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Lys Gln Asp Leu Lys Met Ser Gly Val Leu Arg Ala Glu Ala Gln Glu
145 150 155 160
Arg Thr
<210> 3
<211> 729
<212> DNA
<213> 海生细菌副球菌N81106(Paracoccus sp. N81106)
<400> 3
atgagcgcac atgccctgcc caaggcagat ctgaccgcca ccagcctgat cgtctcgggc 60
ggcatcatcg ccgcttggct ggccctgcat gtgcatgcgc tgtggtttct ggacgcagcg 120
gcgcatccca tcctggcgat cgcaaatttc ctggggctga cctggctgtc ggtcggattg 180
ttcatcatcg cgcatgacgc gatgcacggg tcggtggtgc cggggcgtcc gcgcgccaat 240
gcggcgatgg gccagcttgt cctgtggctg tatgccggat tttcgtggcg caagatgatc 300
gtcaagcaca tggcccatca ccgccatgcc ggaaccgacg acgaccccga tttcgaccac 360
ggcggcccgg tccgctggta cgcccgcttc atcggcacct atttcggctg gcgcgagggg 420
ctgctgctgc ccgtcatcgt gacggtctat gcgctgatcc ttggggatcg ctggatgtac 480
gtggtcttct ggccgctgcc gtcgatcctg gcgtcgatcc agctgttcgt gttcggcacc 540
tggctgccgc accgccccgg ccacgacgcg ttcccggacc gccacaatgc gcggtcgtcg 600
cggatcagcg accccgtgtc gctgctgacc tgctttcact ttggcggtta tcatcacgaa 660
caccacctgc acccgacggt gccgtggtgg cgcctgccca gcacccgcac caagggggac 720
accgcatga 729
<210> 4
<211> 242
<212> PRT
<213> 海生细菌副球菌N81106(Paracoccus sp. N81106)
<400> 4
Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu
1 5 10 15
Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His
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Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Ile Ala
35 40 45
Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala
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His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn
65 70 75 80
Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp
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Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr
100 105 110
Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala
115 120 125
Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro
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Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr
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Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe
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Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro
180 185 190
Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu
195 200 205
Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His
210 215 220
Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp
225 230 235 240
Thr Ala
<210> 5
<211> 39
<212> DNA
<213> 人工序列(Artificial)
<400> 5
acaacaccag atcactcacc atggatgagc gcacatgcc 39
<210> 6
<211> 39
<212> DNA
<213> 人工序列(Artificial)
<400> 6
atcccggtcg gcatctacga tatctcatgc ggtgtcccc 39
<210> 7
<211> 36
<212> DNA
<213> 人工序列(Artificial)
<400> 7
atacattata cgaacggtac ctgtacagtg accggt 36
<210> 8
<211> 36
<212> DNA
<213> 人工序列(Artificial)
<400> 8
ctgatccaag ctcaagctaa gcttgcatgc ctgcag 36
Claims (1)
1.一株产虾青素的红冬孢酵母基因工程菌株,其含有核苷酸序列如SEQ ID NO:1所示的β-胡萝卜素羟化酶基因crtZ、核苷酸序列如SEQ ID NO:3所示的β-胡萝卜素酮化酶基因crtW。
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