CN116426549A - 聚酮-氨基酸化合物及合成基因簇、合成方法与应用 - Google Patents
聚酮-氨基酸化合物及合成基因簇、合成方法与应用 Download PDFInfo
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Abstract
本发明公开聚酮‑氨基酸化合物生物合成基因簇,所述合成基因簇来源于真菌Akanthomyces sp.FJNU 188,包含聚酮合酶‑非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;利用所述聚酮‑氨基酸化合物生物合成基因簇通过异源表达合成聚酮‑氨基酸化合物;聚酮‑氨基酸化合物可用于农业除草中。
Description
技术领域
本发明属于基因工程技术领域,具体涉及聚酮-氨基酸化合物及合成基因簇、合成方法与应用。
背景技术
农业生产的持续增长是应对世界人口的不断增加的必要条件。农药(包括除草剂、杀菌剂和杀虫剂等)的使用为粮食增产和安全提供了重要的保障,是现代农业生产的重要内容。全球农药市场中份额最大的除草剂占45%。除草剂的广泛使用不仅带来了粮食产量和生产效率提高,也导致了杂草的抗性的不断出现和增加,因此,开发新的除草剂成为农业生产的重大需求。
随着基因组测序技术的不断进步和越来越多的微生物基因组序列的公布,我们发现微生物基因组中存在大量尚未解析的次级代谢产物生物合成基因簇。微生物往往通过其合成的次级代谢产物去杀死或抑制竞争对手,从而获得更好的生存空间和生长营养等。如果自身也同样存在这些化合物的作用靶点,那么微生物一定需要自我抗性机制才能免受自身所产生的化合物的毒性。天然产物生物合成基因簇中存在的看家基因的拷贝可能就是一种抗性基因,是化合物的作用靶点蛋白的同工酶,它可以在靶点蛋白受到抑制的情况下行使功能,实现自我抗性的作用。这样,通过生物合成基因簇的抗性基因可以得到其合成的化合物的活性功能。微生物的次级代谢产物合成是受严谨调控的,在实验室培养条件下,多数的基因簇是沉默不表达的,利用异源表达等方法去挖掘这些基因簇,通过强制激活基因簇中的“沉默”基因再结合传统分离手段,成为开发新的天然产物的重要手段。
聚酮化合物是天然产物最多的类群之一,因其巨大的药用价值而被广泛应用于医用、兽用和农用等领域。例如抗生素红霉素、抗寄生虫抗生素阿维菌素等。聚酮化合物是有聚酮合酶(PKS)催化形成的,以模块形式组装成的基因簇决定了聚酮化合物结构从而决定了其生物活性,因此一个新化合物的基因簇对其后续研究有着极为重要的意义。
发明内容
本发明提供一种新化合物,聚酮-氨基酸化合物及合成基因簇、合成方法,聚酮-氨基酸化合物能够用于农业除草。
本发明的技术方案如下:
本发明提供聚酮-氨基酸化合物生物合成基因簇,所述合成基因簇来源于真菌Akanthomyces sp.FJNU 188,包含聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;其中,
所述聚酮合酶-非核糖体肽合成酶基因fuaA的核苷酸序列如SEQ ID No.1所示,编码氨基酸序列为SEQ ID NO.5的聚酮合酶-非核糖体肽合成酶;
所述烯酮还原酶基因fuaB的核苷酸序列如SEQ ID No.2所示,编码氨基酸序列为SEQ ID NO.6的烯酮还原酶;
所述环化酶基因fuaC的核苷酸序列如SEQ ID No.3所示,编码氨基酸序列为SEQID NO.7的环化酶;
所述乙酰羟酸合酶基因fuaD的核苷酸序列如SEQ ID No.4所示,编码氨基酸序列为SEQ ID NO.8的乙酰羟酸合酶;
其中,本发明的真菌Akanthomyces sp.FJNU 188菌株保藏于中国普通微生物菌种保藏管理中心,保藏编号为CGMCC 40319。
第二方面本发明提供一种基因工程菌,含有所述聚酮-氨基酸化合物生物合成基因簇。
此外,本发明提供聚酮-氨基酸化合物,利用所述聚酮-氨基酸化合物生物合成基因簇或所述基因工程菌通过异源表达合成;所述聚酮-氨基酸化合物的结构式如下:
本发明还提供聚酮-氨基酸化合物的合成方法,包括以下步骤:
(1)异源表达菌株的构建:利用PCR技术,以真菌Akanthomyces sp.FJNU188基因组DNA为模板,分别用引物扩增聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;以pYTP质粒为模板,用引物扩增出amyB启动子,以并用Gibson组装方法,将fuaA、amyB启动子和fuaB克隆到表达载体pYTU上构建出表达质粒pYC37、将fuaC克隆到表达载体pYTR上构建出表达质粒pYC38,将fuaD克隆到表达载体pYTP上构建出表达质粒pYC39;用PEG介导的方法将表达质粒pYC37、pYC38和pYC39共同转化至构巢曲霉Aspegillus nidulans A1145中,构建出异源表达菌株AN-fua;
(2)构巢曲霉异源表达菌株的培养和发酵:将经过步骤(1)构建的异源表达菌株AN-fua接种到种子培养基培养,再转移到发酵培养基中发酵培养得到发酵液;
(3)化合物产物分离纯化:收集发酵4天的发酵液,用等体积的萃取液萃取多遍,合并萃取液,用减压浓缩的方法得到粗提取物,然后用硅胶柱层析初步分离,富集目标组分,然后在HPLC上通过半制备柱进行纯化,获得单体化合物聚酮-氨基酸。
优选的,所述步骤(1)中扩增聚酮合酶-非核糖体肽合成酶基因fuaA的引物对如SEQ ID NO.9-12所示,扩增烯酮还原酶基因fuaB的引物对如SEQ ID NO.13-14所示、扩增环化酶基因fuaC的引物对如SEQ ID NO.15-16所示,扩增乙酰羟酸合酶基因fuaD的引物对如SEQ ID NO.17-18所示,扩增amyB启动子的引物对如SEQ ID NO.19-20所示。
优选的,所述步骤(2)中种子培养基为CD培养基,其组成包括质量分数分别为1%Glucose、5%的20x Nitrate salts和0.1%的Trace elements;发酵培养基为CD-ST培养基,其组成包括质量分数分别为2%的Starch、2%的Tryptone、5%的20x Nitrate salts和0.1%的Trace elements。
优选的,所述20x Nitrate包含浓度分别为120g/L的NaNO3、10.4g/L的KCl、10.4g/L的MgSO4·7H2O和30.4g/L的KH2PO4;所述Trace elements包含浓度分别为22.0g/L的ZnSO4·7H2O、11.0g/L的H3BO3、5.0g/L的MnCl2·4H2O、1.6g/L的FeSO4·7H2O、1.6g/L的CoCl2·5H2O、1.6g/L的CuSO4·5H2O、1.11g/L的(NH4)6Mo7O24·4H2O。
第四方面本发明提供的聚酮-氨基酸化合物能够应用在农业除草中。
第五方面本发明提供一种除草剂,包括所述聚酮-氨基酸化合物。
本发明的有益效果在于:本发明公开的聚酮-氨基酸化合物在文献中未见报道,是首次利用包含聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD的生物合成基因簇进行异源表达合成聚酮-氨基酸化合物,实验证明本发明公开的聚酮-氨基酸化合物能够应用在农业除草中,聚酮-氨基酸化合物具有抑制植物生长的作用,对于生物除草剂的开发具有借鉴意义。
附图说明
图1为实施例1中聚酮-氨基酸化合物生物合成基因簇的示意图;
图2为实施例2中聚酮-氨基酸化合物异源表达菌株的构建所用的质粒pYC37、pYC38和pYC39的示意图;
图3为实施例3中异源表达菌株发酵和代谢产物的LC-MS检测图;
图4至图8为本发明合成的聚酮-氨基酸化合物的核磁图谱;
图9为本发明聚酮-氨基酸化合物对拟南芥的生长抑制活性示意图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明做进一步的详细说明。
一般性说明:如下实施例所涉及的pYTP质粒、构巢曲霉Aspegillus nidulansA1145(需要体现微生物出处,购买的话要有购买渠道,在文献中公开的要说明文献出处),实施例中的实验方法及试剂如无特殊说明,均为本领域常规方法与市售试剂。
实施例1
一种聚酮-氨基酸化合物生物合成基因簇,利用许多除草剂靶点的乙酰羟酸合酶氨基酸序列,从真菌Akanthomyces sp.FJNU 188基因组中找到两个可能的乙酰羟酸合酶基因,其中一个在聚酮合酶-非核糖体肽合成酶基因附近,通过用Blast比对GeneBank数据库,对这个基因簇中的基因功能进行了预测;
所述聚酮-氨基酸化合物生物合成基因簇包含聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD,如图1所示;
聚酮合酶-非核糖体肽合成酶基因fuaA的核苷酸序列如SEQ ID No.1所示,编码氨基酸序列为SEQ ID NO.5的聚酮合酶-非核糖体肽合成酶;
烯酮还原酶基因fuaB的核苷酸序列如SEQ ID No.2所示,编码氨基酸序列为SEQID NO.6的烯酮还原酶;
环化酶基因fuaC的核苷酸序列如SEQ ID No.3所示,编码氨基酸序列为SEQ IDNO.7的环化酶;
乙酰羟酸合酶基因fuaD的核苷酸序列如SEQ ID No.4所示,编码氨基酸序列为SEQID NO.8的乙酰羟酸合酶。
实施例2
一种基因工程菌,含有实施例1中的聚酮-氨基酸化合物生物合成基因簇;所述基因工程菌的构建以Akanthomyces sp.FJNU 188基因组为模板,UAF1/R1和UAF2/R2为引物,利用PCR扩增出包含完整聚酮合酶-非核糖体肽合成酶基因fuaA基因的具有66个碱基对重叠的两个片段,UBF/R为引物,扩增出烯酮还原酶fuaB基因;利用UAmyBF/R为引物,以表达载体pYTP为模板,扩增出amyB启动子,收集4个PCR产物和PacI,SwaI双酶切的pYTU载体片段,利用Gibson组装成表达质粒pYC37;以Akanthomyces sp.FJNU 188基因组为模板,以RCF/R为引物,利用PCR扩增出环化酶fuaC基因,收集PCR产物和PacI,SwaI双酶切的pYTR载体片段,利用Gibson组装成表达质粒pYC38;以Akanthomyces sp.FJNU 188基因组为模板,以PDF/R为引物、利用PCR扩增出乙酰羟酸合酶fuaD基因,收集PCR产物和PacI,SwaI双酶切的pYTP载体片段,利用Gibson组装成表达质粒pYC39(表达质粒pYC37、pYC38和pYC39如图2所示);用PEG介导的方法将表达质粒pYC37、pYC38和pYC39共同转化至构巢曲霉Aspegillusnidulans A1145中,构建出异源表达基因工程菌株AN-fua;
其中,上述引物UAF1/UAR1、UAF2/UAR2、UBF/UBR、RCF/RCR、PDF/PDR、UAmyBF/UAmyBR序列如表1所示;
表1引物序列
将异源表达基因工程菌株AN-fua接种于5mL液体发酵培养基CD-ST(2%Starch,2% Tryptone,5%20x Nitrate salts和0.1% Trace elements)中,25℃摇瓶培养4天,收集500μL发酵4天的发酵液,用等体积的萃取液(89%乙酸乙酯、10%甲醇和1%醋酸)萃取,用减压浓缩的方法去除萃取液,然后用100μL甲醇溶解萃取物用于液质联用分析;分析方法如下:所用仪器为Thermo U3000HPLC-LCQ fleet质谱,配phenomenex Luna 5μm C18(2),150x 2.1mm色谱柱,液相柱温控制为30℃,检测波长设置为200-800nm。液相色谱流速为0.25mL/min,流动相A为含0.1%甲酸的超纯水,流动相B为含0.1%甲酸的HPLC级乙腈,0-30min过程中B相由5%线性升至95%,质谱仪离子源设定参数为:喷雾器压力为35psi,干燥气流为35ml/min,温度为350℃,扫描模式为正负离子连续全扫描,扫描范围为m/z=120-1200;由液质联用的结果图3中显色,与对照组(构巢曲霉含三个空载体pYTU,pYTR,pYTP)相比,AN-fua的发酵液在min中出现一化合物峰,其加氢离子峰为404。
实施例3
聚酮-氨基酸化合物,利用所述聚酮-氨基酸化合物生物合成基因簇或所述基因工程菌通过异源表达合成;通过NMR确定合成的单体化合物的化学结构(如图4至8所示),表2中列出化合物的核磁数据归属;
表2聚酮-氨基酸化合物核磁数据归属(1H for 600MHz and 13C for 150MHz inCDCl3)
实施例4
聚酮-氨基酸化合物的合成方法,包括以下步骤:
(1)异源表达菌株的构建:利用PCR技术,以真菌Akanthomyces sp.FJNU188基因组DNA为模板,分别用引物扩增聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;以pYTP质粒为模板,用引物扩增出amyB启动子,以并用Gibson组装方法,将fuaA、amyB启动子和fuaB克隆到表达载体pYTU上构建出表达质粒pYC37、将fuaC克隆到表达载体pYTR上构建出表达质粒pYC38,将fuaD克隆到表达载体pYTP上构建出表达质粒pYC39;将菌株Aspergillus nidulans A1145接种到PDA培养基上,30℃培养5天,收集孢子,将孢子悬液接种于40mL的PDB液体培养基中,于30℃,250rpm培养约10h;用无菌滤孢器收集菌丝体,用20mL Osmotic buffer(1.2M MgSO4,10mM磷酸氢二钠-磷酸二氢钠缓冲液,pH5.8,)吹洗三遍,将菌丝体转至10mL裂解液(35mg lysing enzymesfrom Trichoderma和20mg Yatalase溶于10mL Osmotic buffer)中,30℃,80r/min酶解,镜检;将酶解液转移到50mL离心管中,加入10mL Trapping buffer(0.6M山梨醇,0.1M Tris-HCl,pH 7.0),4℃,4000rpm离心10min,两种buffer的中间白色絮状物层即为原生质体;将原生质体转移到新的50mL离心管,加入15mL STC buffer(1.2M山梨醇,10mM CaCl2,10mMTris-HCl,pH 7.5)轻轻混匀,4000rpm,4℃离心10min,弃上清;加入适量STC buffer重悬原生质体;在原生质体管中加入4个表达质粒和PEG solution(60%PEG 4000,50mM CaCl2,50mM Tris-HCl,pH 7.5)轻弹混匀,室温放置20min;将混合液涂布于转化平板上(1%Glucose,5%20x Nitrate salts,0.1% Trace elements,0.6M山梨醇,1%琼脂),30℃倒置培养2-3天,长出的菌落即为表达菌株AN-fua;
(2)构巢曲霉异源表达菌株的培养和发酵:将经过步骤(1)构建的异源表达菌株AN-fua接种到种子培养基,25℃摇瓶培养2天,种子培养基为CD培养基,包括质量分数分别为1% Glucose、5%的20x Nitrate salts和0.1%的Trace elements;再将其全部转移到发酵培养基中,25℃摇瓶培养4天得到发酵液,发酵培养基为CD-ST培养基,包括质量分数分别为2%的Starch、2%的Tryptone、5%的20x Nitrate salts和0.1%的Trace elements;
其中,所述20x Nitrate包含浓度分别为120g/L的NaNO3、10.4g/L的KCl、10.4g/L的MgSO4·7H2O和30.4g/L的KH2PO4;所述Trace elements包含浓度分别为22.0g/L的ZnSO4·7H2O、11.0g/L的H3BO3、5.0g/L的MnCl2·4H2O、1.6g/L的FeSO4·7H2O、1.6g/L的CoCl2·5H2O、1.6g/L的CuSO4·5H2O、1.11g/L的(NH4)6Mo7O24·4H2O;
(3)化合物产物分离纯化:收集发酵4天的发酵液,用等体积的萃取液(89%乙酸乙酯、10%甲醇和1%醋酸)萃取多遍,合并萃取液,用减压浓缩的方法得到粗提取物,先用Combiflash制备色谱进行初分离(色谱柱:RediSepRf,80g Silica柱),用甲醇溶解样品,称取适量硅藻土拌样(样品与硅藻土比例为1:2),减压旋转蒸干,上样,洗脱条件为:起始用石油醚-乙酸乙酯体系,从0%乙酸乙酯梯度到90%乙酸乙酯,时间40min;然后用二氯甲烷-甲醇体系,从0%甲醇梯度到50%甲醇,时间30min。全程流速45ml/min。收集方式选择全收集。隔管取样液质检测后将含目标产物的管合并减压浓缩,用甲醇溶解,然后在HPLC上通过半制备柱phenomenex Luna 5μm C18(2),250x 10mm进行纯化,获得聚酮-氨基酸单体化合物。
实施例5
将聚酮-氨基酸化合物和阳性对照草铵膦分别加入到含有0.8%琼脂的MS培养基中,相应培养基倒入平板中使其冷却;拟南芥种子在经过灭菌后将其播种到带有培养基的平板中;经过两天4℃储存以模拟春化过程后,拟南芥植株在培养箱中垂直生长,温度为22℃,光照时间为16小时/8小时,7天后进行观察,结果见图9;结果显示,聚酮-氨基酸化合物具有较好的抑制植物生长作用,可以用于农业除草剂方面的应用。
提供上述实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
Claims (10)
1.聚酮-氨基酸化合物生物合成基因簇,其特征在于:所述合成基因簇来源于真菌Akanthomyces sp.FJNU 188,包含聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;其中,
所述聚酮合酶-非核糖体肽合成酶基因fuaA的核苷酸序列如SEQ ID No.1所示,编码氨基酸序列为SEQ ID NO.5的聚酮合酶-非核糖体肽合成酶;
所述烯酮还原酶基因fuaB的核苷酸序列如SEQ ID No.2所示,编码氨基酸序列为SEQID NO.6的烯酮还原酶;
所述环化酶基因fuaC的核苷酸序列如SEQ ID No.3所示,编码氨基酸序列为SEQ IDNO.7的环化酶;
所述乙酰羟酸合酶基因fuaD的核苷酸序列如SEQ ID No.4所示,编码氨基酸序列为SEQID NO.8的乙酰羟酸合酶。
2.一种基因工程菌,其特征在于,含有如权利要求1所述聚酮-氨基酸化合物生物合成基因簇。
4.聚酮-氨基酸化合物的合成方法,其特征在于,包括以下步骤:
(1)异源表达菌株的构建:利用PCR技术,以真菌Akanthomyces sp.FJNU 188基因组DNA为模板,分别用引物扩增聚酮合酶-非核糖体肽合成酶基因fuaA、烯酮还原酶基因fuaB、环化酶基因fuaC和乙酰羟酸合酶基因fuaD;以pYTP质粒为模板,用引物扩增出amyB启动子,以并用Gibson组装方法,将fuaA、amyB启动子和fuaB克隆到表达载体pYTU上构建出表达质粒pYC37、将fuaC克隆到表达载体pYTR上构建出表达质粒pYC38,将fuaD克隆到表达载体pYTP上构建出表达质粒pYC39;用PEG介导的方法将表达质粒pYC37、pYC38和pYC39共同转化至构巢曲霉Aspegillus nidulans A1145中,构建出异源表达菌株AN-fua;
(2)构巢曲霉异源表达菌株的培养和发酵:将经过步骤(1)构建的异源表达菌株AN-fua接种到种子培养基培养,再转移到发酵培养基中发酵培养得到发酵液;
(3)化合物产物分离纯化:收集发酵4天的发酵液,用等体积的萃取液萃取多遍,合并萃取液,用减压浓缩的方法得到粗提取物,然后用硅胶柱层析初步分离,富集目标组分,然后在HPLC上通过半制备柱进行纯化,获得单体化合物聚酮-氨基酸。
5.如权利要求4所述的聚酮-氨基酸化合物的合成方法,其特征在于,所述步骤(1)中扩增聚酮合酶-非核糖体肽合成酶基因fuaA的引物对如SEQ IDNO.9-12所示,扩增烯酮还原酶基因fuaB的引物对如SEQ ID NO.13-14所示、扩增环化酶基因fuaC的引物对如SEQ IDNO.15-16所示,扩增乙酰羟酸合酶基因fuaD的引物对如SEQ ID NO.17-18所示,扩增amyB启动子的引物对如SEQ ID NO.19-20所示。
6.如权利要求4所述的聚酮-氨基酸化合物的合成方法,其特征在于,所述步骤(2)中种子培养基为CD培养基,其组成包括质量分数分别为1%的Glucose、5%的20x Nitratesalts和0.1%的Trace elements;发酵培养基为CD-ST培养基,其组成包括质量分数分别为2%的Starch、2%的Tryptone、5%的20xNitrate salts和0.1%的Trace elements。
7.如权利要求6所述的聚酮-氨基酸化合物的合成方法,其特征在于,所述20x Nitrate包含浓度分别为120g/L的NaNO3、10.4g/L的KCl、10.4g/L的MgSO4·7H2O和30.4g/L的KH2PO4;所述Trace elements包含浓度分别为22.0g/L的ZnSO4·7H2O、11.0g/L的H3BO3、5.0g/L的MnCl2·4H2O、1.6g/L的FeSO4·7H2O、
8.1.6g/L的CoCl2·5H2O、1.6g/L的CuSO4·5H2O、1.11g/L的(NH4)6Mo7O24·4H2O。
9.如权利要求3所述的聚酮-氨基酸化合物在农业除草中的应用。
10.一种除草剂,其特征在于,包括如权利要求3的聚酮-氨基酸化合物。
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