CN116286767B - β-石竹烯合成酶及基因与菌株和应用 - Google Patents
β-石竹烯合成酶及基因与菌株和应用 Download PDFInfo
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Abstract
本发明公开了β‑石竹烯合成酶及基因与菌株和应用。本发明首次发现了橡胶树Hevea brasiliensis来源的氨基酸序列如SEQ ID NO.1所示的酶为β‑石竹烯的合成酶,其或其编码基因或含有其编码基因的重组质粒、重组菌株可用于合成或生产β‑石竹烯。一种β‑石竹烯高产菌株为在过表达甲羟戊酸途径中的一个或多个基因以及法尼烯焦磷酸合酶基因的微生物中导入β‑石竹烯合成酶的编码基因获得,本发明提供的β‑石竹烯高产菌株的发酵罐(5L钢罐)产量高达6.2g/L。制备得到的β‑石竹烯可用于制备香料、香精及医药中间体。
Description
技术领域
本发明属于基因工程技术领域,具体涉及β-石竹烯合成酶及基因与菌株和应用。
背景技术
β-石竹烯属于萜类家族一员,是一种倍半萜,具有辛香、木香、柑橘香、樟脑香、温和的丁香香气,主要存在于植物人参、丁香花等植物中。β-石竹烯被国家GB2760-1996批准为允许使用的食品香料。目前主要用于调配丁香、胡椒、肉豆蔻、柑橘、药草等食用香精,也可用于合成如乙酰基石竹烯等更有价值的香料。除了用作香料物质外,β-石竹烯的生物活性研究表明,其具有抗癌、抗炎、抗氧化及保护神经等多种生理活性。β-石竹烯不仅自身具有抗癌活性而且还可以通过增加细胞膜的通透性来显著提高其他化疗药物的抗癌活性。例如,β-石竹烯可以增加紫杉醇对细胞膜的通透性,使其对结肠腺癌细胞DLD-1的抑制活性提高10倍。β-石竹烯目前主要生产方式是来自于丁香油经真空分馏获得,生物技术合成β-石竹烯还处于起步阶段,产量还较低,远达不到工业化水平,研究β-石竹烯的生物合成具有重要研究意义,有进一步开发的价值。
发明内容
本发明的目的在于,针对现有技术的上述不足,提供了β-石竹烯合成酶及基因与菌株和应用。
为实现上述目的,本发明采用如下的技术方案:
本发明的第一目的是提供一种β-石竹烯合成酶HbBaS,所述β-石竹烯合成酶HbBaS的氨基酸序列如SEQ ID NO.1所示。
本发明的第二目的是提供一种β-石竹烯合成酶基因HbBACS在合成或生产β-石竹烯中的应用,所述β-石竹烯合成酶基因HbBACS的碱基序列与SEQ ID NO:2所示的碱基序列具有95%以上的同一性。
进一步的,所述β-石竹烯合成酶基因HbBACS的碱基序列如SEQ ID NO:2所示。
本发明的第三目的是提供一种重组表达载体,包括上述的β-石竹烯合成酶基因HbBACS。
本发明的第四目的是提供一种上述的重组表达载体的构建方法,包括如下步骤:在权利要求2-3中任一项所述的基因HbBACS分别加上酶切位点,然后分别用限制性内切酶Sal I和Xho I双酶切,得到具有粘性末端的基因HbBACS片段;将酿酒酵母商用表达载体pRS426分别用限制性内切酶Pme I和Not I双酶切,得到具有粘性末端的载体pRS426片段;将所述具有粘性末端的基因HbBACS片段和所述具有粘性末端的载体pRS426片段通过T4DNA连接酶连接,获得β-石竹烯合成酶重组表达载体pHbBACS。
本发明的第五目的是提供一种重组表达菌株,包括上述的重组表达载体。
进一步的,所述重组表达菌株的宿主细胞为酿酒酵母。
本发明的第六目的是提供上述重组表达菌株的制备方法,将β-石竹烯合成酶的重组表达载体pHbBACS通过酶切位点将其线性化;将线性化的重组表达载体pHbBACS导入宿主细胞中,获得重组表达菌株。
本发明的第七目的是提供一种β-石竹烯的生产方法,培养上述的重组表达菌株,从培养物中获得β-石竹烯。
本发明的第八目的是提供上述的β-石竹烯在制备香料或香精中的应用。
与现有技术比较,本发明提供的技术方案带来的有益效果是:
(1)本发明首次发现了橡胶树来源的β-石竹烯合成酶及其编码基因,该酶能够高效地合成产物β-石竹烯。
(2)构建的β-石竹烯高产菌株,摇瓶产量达到126mg/L,发酵罐(5L钢罐)产量达到6.2g/L,为目前报道的最高产量,为β-石竹烯的工业化应用奠定坚实的基础。
附图说明
图1为本发明的JBS1菌株产物的GC-MS检测结果图;
图2为本发明用到的橡胶树胶乳图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例和附图,对本发明的具体实施方式作进一步详细描述。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
Taq DNA聚合酶、限制性内切酶、质粒提取试剂盒和DNA提取试剂盒均购自Takara公司;DNA胶回收试剂盒购自诺维赞公司;同源重组试剂盒购自翊圣公司;表达质粒pRS426购自ATCC公司;特异基因引物对P1/P2由金开瑞公司合成。
β-石竹烯的检测方法:收集两相发酵培养的有机相,以3500rpm离心10分钟以除去细胞碎片,并用正己烷稀释到适当的范围。然后将样品进行气相色谱法质谱法(GC-MS)检测分析,使用TSQ QUANTUM XLS质谱仪(Thermo Fisher Scientific,Thermo,MA,USA),用TR-5MS柱(30m×0.25mm×0.25μm)检测和定量β-石竹烯的浓度。GCMS的程序设定如下:温度最初保持在50℃ 1分钟,然后以15℃/分钟的速度增加到280℃,保持1分钟,然后以20℃/分钟的速度增加到300℃,保持2分钟。化合物通过与标准品比对或通过NIST(NationalInstitute of Standards and Technology)数据库和保留指数的比较确定,标准品β-石竹烯购自Sigma-Aldrich(St.Louis,MO,U.S.A.);
YPD培养基:培养基组分含有2%葡萄糖(国药公司),2%胰蛋白胨(安琪酵母FP318),1%酵母抽提物(安琪酵母FM888)
酵母菌株YZL141,其构建方法记载在Shi Bin et al.,“Systematic MetabolicEngineering of Saccharomyces cerevisiae for Lycopene Overproduction.”Journalof agricultural and food chemistry vol.67,40(2019):11148-11157.doi:10.1021/acs.jafc.9b04519。
酵母菌株JCR27,其构建方法记载在Siemon,Thomas et al.“Semisynthesis ofPlant-Derived Englerin AEnabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.”Journal of the American Chemical Society vol.142,6(2020):2760-2765.doi:10.1021/jacs.9b12940。
橡胶树cDNA:从橡胶树胶乳(如图2所示)中提取的RNA,再反转录成cDNA,该过程通常按照常规条件如《分子克隆实验指南(第四版)》(中文版)由科学出版社出版中所说的条件,或按照制造产商所建议的条件,其为本领域技术人员熟知的实验操作,本发明对此并无限定。
实施例1
(1)重组载体构建
本申请人对橡胶树的基因组进行了分析,发现了一个萜类合酶HbBaS,其氨基酸序列如SEQ ID NO.1所示,其核苷酸序列如SEQ ID NO.2所示。
设计特异基因引物对P1/P2(如表1所示),以橡胶树cDNA为模板,利用Takara公司的Prime STAR MAX PCR酶通过PCR扩增获得特异基因片段,经诺维赞胶回收试剂盒回收片段后,通过翊圣公司同源重组试剂盒,采用同源重组的方法连接到酵母商用表达载体pRS426上(Sal I/Xho I)双酶切),经过测序确认无误后,获得含有该特异基因HbBACS的酵母表达载体,命名为pHbBACS。
表1.
(2)菌株构建
通过PEG/LiAC方法将pHbBACS质粒转化到酵母菌株YZL141感受态中,涂布于SD-URA筛选平板上,培养3d后,利用P1和P2引物,使用诺唯赞PCR试剂进行菌落PCR验证,将阳性菌命名为YHbBACS。菌株YHbBACS是在酿酒酵母CEN.PK2-1D基础上,首先加强了MVA途径,过表达了MVA途径的限速酶tHMG1,并转入了特异基因HbBACS的质粒。
同时利用PmeI内切酶酶切pHbBACS回收含有HbBACS基因片段,通过PEG/LiAC方法将该片段导入到酵母菌株JCR27感受态中,进行酵母菌落PCR验证后,将阳性菌命名为JBS1。菌株JBS1相较于CEN.PK2-1D菌株,加强了MVA途径,过表达整个MVA途径,在此基础上,又过表达了HbBACS基因,菌株JBS1中各基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、HbBACS=2、2、2、2、2、2、2、2、1。
(3)摇瓶发酵
将YHbBACS菌株接种于50mL补充1%半乳糖的YPD培养基中,加入1% IPM,30℃培养3d后,8000rpm离心10min收集覆盖层的有机油相,并用正己烷稀释到适当的范围进行GC-MS检测。检测如图1所示,主峰通过和β-石竹烯标准品保留时间及MS数据对比,可以确定菌株YHbBACS可以合成β-石竹烯,说明基因HbBACS为β-石竹烯合成酶基因,摇瓶产量为18mg/L;小峰产物经过搜NIST数据库比对,产物为α-葎草烯。
JBS1菌株采用类似的方法,添加10%肉豆蔻酸异丙酯(IPM)覆盖后发酵72h,离心后取有机油相,并用正己烷稀释到适当的范围进行GC-MS检测。倍半萜产物β-石竹烯的产量从菌株YHbBACS的18mg/L增加至菌株JBS1的126mg/L,产量增加约7倍。
参照文献(van Hoek,P.;de Hulster,E.;van Di jken,J.P.;Pronk,J.T.Fermentative capacity in high-cell-density fed-batch cultures of baker’syeast.Biotechnol.Bioeng.2000,68,517-523.)中所记载的发酵培养基,对所构建的菌株JBS1进行分批补料发酵,在发酵过程中添加覆盖剂以实现原位萃取,覆盖剂为IPM肉豆蔻酸异丙酯。发酵过程控制溶氧在20%以上,pH为5,葡萄糖浓度为1-2g/L,乙醇浓度为5g/L以下。最终在发酵罐(5L发酵罐)上,倍半萜产物β-石竹烯产量达到了6.2g/L。
实施例2
为了评价β-石竹烯作为香料和调味剂的潜在价值,由品香师分析了该分子中的香味,β-石竹烯具有辛香、木香、柑橘香、樟脑香、温和的丁香香气。当β-石竹烯被加热时,伴随着增强的气味产生白烟,这表明它有潜力用作香料成分和食品调味剂。
本领域技术人员知晓,β-石竹烯已被国标GB 2670—1996规定为允许使用的食用香料,其可用于辛香香型的日化及食用香精的配香,以及用于烟用香精的协调,β-石竹烯对增加焦甜香气也具有较好的效果。
实施例3
由本发明中的β-石竹烯新合成酶基因构建的重组酿酒酵母发酵收集得到的有机覆盖相,通过精馏的方式,将不同的组分进行区分,纯化所得的β-石竹烯纯度可达95%以上,本发明发酵收集纯化的β-石竹烯可用作医药中间体,作为继续合成其他的药物的原料药,
在不冲突的情况下,本文中上述实施例及实施例中的特征可以相互结合。
以上所述仅为本发明的较佳实施例,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、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1. 一种β-石竹烯合成酶HbBaS,其特征在于:所述β-石竹烯合成酶HbBaS的氨基酸序列如SEQ ID NO.1所示。
2. 一种β-石竹烯合成酶基因HbBACS在合成或生产β-石竹烯中的应用,其特征在于,所述β-石竹烯合成酶基因HbBACS的碱基序列如SEQ ID NO:2所示。
3.一种重组表达载体,其特征在于,包括如权利要求2中所述的基因HbBACS。
4.一种如权利要求3所述的重组表达载体的构建方法,其特征在于,包括如下步骤:在权利要求2中所述的基因HbBACS上通过特异引物对P1/P2引入酶切位点SalI和XhoI,同时将酿酒酵母商用表达载体pRS426用限制性内切酶SalI和XhoI双酶切,得到具有粘性末端的载体pRS426片段;将引入酶切位点的基因HbBACS片段和所述具有粘性末端的载体pRS426片段通过同源重组的方法进行连接,获得β-石竹烯合成酶重组表达载体pHbBACS;
所述引物P1的序列为GGGCCCGGGCGTCGACATGGCATTTCAAGATTCAGCTTTTTTC;
所述引物P2的序列为GTACCAAGCTTACTCGAGTCAGAGCACAGGGTCCTTAA。
5.一种重组表达菌株,其特征在于,包括如权利要求3所述的重组表达载体。
6.如权利要求5所述的重组表达菌株,其特征在于,所述重组表达菌株的宿主细胞为酿酒酵母。
7.一种如权利要求5或6所述的重组表达菌株的制备方法,其特征在于,包括如下步骤:将如权利要求3所述的重组表达载体通过酶切位点将其线性化;将线性化的重组表达载体导入宿主细胞中,获得重组表达菌株。
8.一种β-石竹烯的生产方法,其特征在于:培养如权利要求5或6所述的重组表达菌株,从培养物中获得β-石竹烯。
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