CN113832123A - 聚酮合酶Preu3-ΔCMeT及其在制备苔色酸中的应用 - Google Patents
聚酮合酶Preu3-ΔCMeT及其在制备苔色酸中的应用 Download PDFInfo
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Abstract
本发明涉及一种聚酮合酶Preu3‑△CMeT及其在制备苔色酸中的应用,属于分子生物学与生物化学技术领域。本发明所述聚酮合酶Preu3‑∆CMeT的氨基酸序列如SEQ ID NO.1所示,其是以光黑壳属真菌来源的聚酮合酶Preu3为材料,基于组合生物合成技术敲除其CMeT结构域构建而成;将其转入酿酒酵母,成功获得可高效生产苔色酸的突变株;并以制备的苔色酸为对象,研究了其对临床耐药细菌以及农作物病原真菌的抑制活性,发现其对耐碳青霉烯铜绿假单胞菌有很强的拮抗作用,MIC为12.5μg/mL。本发明极大地丰富了苔色酸的生产来源,对于拓展其衍生化途径,研发新型抗菌剂有重要的科学价值和应用前景。
Description
技术领域
本发明属于分子生物学与生物化学技术领域,具体涉及一种新型“非天然”聚酮合酶Preu3-ΔCMeT及其在制备苔色酸中的应用。
背景技术
真菌聚酮化合物(polyketides,PKs)是一大类重要的天然产物类群,具有化学结构和生物活性多样性,是新型药物先导化合物发现的优质资源库。代表性的真菌PKs有降低胆固醇功效的洛伐他汀(lovastatin)、真菌抑制剂灰黄霉素(griseofulvin)、细菌抑制剂利福霉素(rifamycin)、免疫抑制剂霉酚酸(mycophenolic acid)、肌动蛋白和血管生成抑制剂细胞松弛素E(cytochalasin E)等。PKs生物合成的关键酶——真菌聚酮合酶(polyketide synthase,PKS)为迭代(iterative)I型PKS,结构域通常包括:起始单元ACP转酰酶(starter unit ACP transacylase,SAT)、β-酮酰基合成酶(β-ketoacylsynthase,KS)、酰基转移酶(acyltransferase,AT)、产物模板(product template,PT)、酰基载体蛋白(acyl carrier protein,ACP)、酮基还原酶(ketoreductase,KR)、脱水酶(dehydratase,DH)、烯基还原酶(enoylreductase,ER)、甲基转移酶(methyl transferase,CMeT)以及硫酯酶(thioesterase,TE)或还原酶(reductase,R)。其中,CMeT负责在聚酮碳链的延伸过程中引入甲基。
苔色酸(orsellinic acid,结构见图1)是一种芳香聚酮小分子化合物,其是在聚酮合酶催化下,由一分子乙酰辅酶A与三分子丙二酰辅酶A逐步缩合而成。目前,已在草本植物、地衣、真菌和细菌中分离鉴定了200余种苔色酸衍生物,包括单体和聚合物。经研究报道,苔色酸及其衍生物具有抗炎、抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性,故其可作为食品与医药行业天然抗菌剂、抗氧化剂及新型药物等的潜在生物资源。但目前,市售苔色酸(CAS:480-64-8)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高,故导致其在食品、医药等领域应用受限。因此如何克服苔色酸现有制备技术的不足是目前微生物化学技术领域亟需解决的问题。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种新型“非天然”聚酮合酶Preu3-ΔCMeT的构建及其在制备苔色酸中的应用。本发明以光黑壳属真菌来源的聚酮合酶Preu3(合成3-甲基苔色酸)为材料,基于组合生物合成技术敲除其CMeT结构域构建了新型“非天然”聚酮合酶Preu3-ΔCMeT;将其转入酿酒酵母,成功获得可高效生产苔色酸的突变株。本发明极大地丰富了苔色酸的生产来源,对于拓展其衍生化途径,研发新型抗菌剂有重要的科学价值和应用前景。
为实现上述目的,本发明采用的技术方案如下:
聚酮合酶Preu3-ΔCMeT,其特征在于,所述聚酮合酶Preu3-ΔCMeT的氨基酸序列如SEQ ID NO.1所示。
编码权利要求1所述聚酮合酶Preu3-ΔCMeT的基因。
进一步,优选的是,所述基因的编码序列是SEQ ID NO.2所示的核苷酸序列。
本发明同时提供含有上述基因的重组载体。
本发明并提供一种用上述重组载体转化得到的重组基因工程菌。
本发明再提供权利要求1所述的聚酮合酶Preu3-ΔCMeT在制备苔色酸中的应用。
本发明还提供一株可高效生产苔色酸的酵母突变株的构建方法,包括如下步骤:
采用LiAc/PEG4000介导的转化法利用权利要求4所述的重组载体转化酿酒酵母,将转化液均匀涂布于SC-Ura固体平板上,30℃培养箱中培养,即得到酵母突变株。
本发明另外提供苔色酸的制备方法,采用上述构建方法构建的酵母突变株,其特征在于,包括如下步骤:
步骤(1),将酵母突变株接种于SC-Ura液体培养基中,30℃,摇床培养;培养的第2d,加入1%YPD培养基继续培养3-4d,得到发酵液;
其中,1%YPD培养基和SC-Ura液体培养基体积相同;
步骤(2),对步骤(1)培养得到的发酵液,用等体积的乙酸乙酯萃取,对萃取液浓缩,得到粗浸膏;
步骤(3),对步骤(2)得到的粗浸膏提纯,得到苔色酸。
进一步,优选的是,步骤(3)中,对步骤(2)得到的粗浸膏进行提纯,提纯方法为:将粗浸膏用甲醇溶解后,通过ODS C18反相硅胶柱层析法,选用甲醇-水为流动相对粗浸膏进行梯度洗脱,共得到五个组分段Fr.A-Fr.E;目标代谢物苔色酸集中在Fr.E组分中,将Fr.E组分通过高效液相制备得到苔色酸;
其中,梯度洗脱流动相采用的甲醇:水的体积比依次为5:95、10:90、15:85、20:80、25:75,各梯度洗脱所用的流动相总体积均为SC-Ura液体培养基体积的十分之一。
本发明最后提供上述苔色酸的制备方法制得的苔色酸在制备抗农作物病原真菌药物或临床耐药细菌药物中的应用,其特征在于,所述的农作物病原真菌为苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌;所述的临床耐药细菌为耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌。
苔色酸及其衍生物具有抗炎、抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性,故其可作为食品与医药行业天然抗菌剂、抗氧化剂及新型药物等的潜在生物资源。但目前,市售苔色酸(CAS:480-64-8)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高,故导致其在食品、医药等领域应用受限。
发明人前期从光黑壳属真菌(Preussia isomera,GenBank编号为MK300824.1)中克隆到与聚酮分子3-甲基苔色酸(3-methylorsellinic acid,结构见图1)合成相关的聚酮合酶Preu3(ZL202110910678.3),其结构域组成为SAT-KS-AT-PT-ACP-CMeT-TE。从化学结构来说,3-甲基苔色酸为苔色酸在3位C上甲基化的产物,也即是聚酮合酶Preu3的CMeT结构域在3-甲基苔色酸合成过程中在其3位C上引入了甲基。因此,发明人拟以聚酮合酶Preu3为对象,基于组合生物合成技术敲除其CMeT结构域构建可高产苔色酸的新型“非天然”聚酮合酶。
在本发明中,我们首次以光黑壳属真菌来源的聚酮合酶Preu3(合成3-甲基苔色酸)为材料,基于组合生物合成技术敲除其CMeT结构域构建了新型“非天然”聚酮合酶Preu3-ΔCMeT;将其转入酿酒酵母,成功获得可高效生产苔色酸的突变株;并以制备的苔色酸为对象,研究了其对八种临床耐药细菌以及七种农作物病原真菌的抑制活性。研究发现,苔色酸对耐碳青霉烯铜绿假单胞菌一种临床耐药细菌以及苹果轮纹病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌四种农作物病原真菌均有不同程度的抑制作用,其中对耐碳青霉烯铜绿假单胞菌有很强的拮抗作用(MIC,12.5μg/mL)。本发明极大地丰富了苔色酸的生产来源,对于拓展其衍生化途径,研发新型抗菌剂有重要的科学价值和应用前景。
本发明与现有技术相比,其有益效果为:
1.目前,市售苔色酸(CAS:480-64-8)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高。本发明基于组合生物合成技术构建了新型“非天然”聚酮合酶Preu3-ΔCMeT,并基于异源表达技术,成功获得可高效生产苔色酸的酵母突变株(苔色酸产量约0.1g/L),提供了新的微生物发酵途径来生产苔色酸,工艺简单,产量高,且绿色环保,具有可观的应用前景。
2.本发明同时也首次研究了苔色酸对七种农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)及八种临床耐药细菌(耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌)的抑制活性。研究发现,苔色酸对耐碳青霉烯铜绿假单胞菌有很强的拮抗作用(MIC,12.5μg/mL),有望研发新型抗生素。
附图说明
图1为苔色酸(orsellinic acid)与3-甲基苔色酸(3-methylorsellinic acid)的化学结构式;其中,(a)为苔色酸;(b)为3-甲基苔色酸;
图2为聚酮合酶Preu3-ΔCMeT及其异源表达载体YEpADH2p-URA-Preu3-ΔCMeT;其中,(a)为聚酮合酶Preu3-ΔCMeT的结构域组成;(b)为异源表达载体YEpADH2p-URA-Preu3-ΔCMeT的质粒图谱;
图3为聚酮合酶Preu3-ΔCMeT异源表达载体YEpADH2p-URA-Preu3-ΔCMeT的酶切验证图;其中,M,Trans 2K plus marker;Line 1,载体YEpADH2p-URA-Preu3-ΔCMeT;
图4为聚酮合酶Preu3-ΔCMeT酿酒酵母异源表达突变株的代谢产物分析;其中,(a)为空载体酿酒酵母;(b)为聚酮合酶Preu3酿酒酵母突变株;(c)为聚酮合酶Preu3-ΔCMeT酿酒酵母突变株;
图5为聚酮合酶Preu3-ΔCMeT酿酒酵母突变株目标代谢产物的紫外吸收图;
图6为聚酮合酶Preu3-ΔCMeT酿酒酵母突变株目标代谢产物的高分辨质谱图;其中,(a)为负离子模式,(b)为正离子模式;
图7为化合物苔色酸的1H核磁光谱图(氘代甲醇,500MHz);
图8为化合物苔色酸的13C核磁光谱图(氘代甲醇,125MHz)。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。
本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,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。所用材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
本发明中我们以光黑壳属真菌(Preussia isomera,GenBank编号为MK300824.1)来源的聚酮合酶Preu3(合成3-甲基苔色酸,ZL202110910678.3)为材料,基于组合生物合成技术敲除其CMeT结构域构建了新型“非天然”聚酮合酶Preu3-ΔCMeT;将其转入酿酒酵母,成功获得可高效生产苔色酸(orsellinic acid,结构见图1)的酵母突变株。本实验的大致步骤为:首先基于组合生物合成及同源重组技术,将Preu3的CMeT结构域进行敲除,构建聚酮合酶Preu3-ΔCMeT,并构建其异源表达载体YEpADH2p-URA-Preu3-ΔCMeT(质粒图谱见图2);然后采用PEG4000/LiAc转化法,将构建的异源表达载体转化酿酒酵母,获得阳性酵母转化株,分析其代谢产物并进行结构鉴定,发现构建的酵母突变株可高产苔色酸(~0.1g/L);最后,以制备的苔色酸为对象,研究了其对七种农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)及八种临床耐药细菌(耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌)的抑制活性(实验结果见表3、4)。具体实验过程如下。
1.聚酮合酶Preu3-ΔCMeT及其异源表达载体YEpADH2p-URA-Preu3-ΔCMeT的构建
1.1聚酮合酶基因preu3-ΔCMeT(结构域组成见图2)的构建:敲除聚酮合酶preu3的CMeT结构域(V1789-G2114,对应碱基G5365-A6342),构建新型聚酮合酶基因preu3-ΔCMeT,其mRNA序列全长6594-bp,如SEQ ID NO.2,克隆引物见表1。
表1:聚酮合酶基因preu3-ΔCMeT的克隆相关引物信息
PCR反应体系配置(100μL):ddH2O 55.5μL;5×Phusion HF buffer 20μL;dNTPs(2.5mmol/L)8μL;正向引物(10pmol/μL)5μL;反向引物(10pmol/μL)5μL;DMSO 3μL;PhusionDNA Ploymerase(2U/μL)1μL;cDNA 2.5μL。PCR程序是:98℃ 3min;98℃ 30s,58℃ 30s,72℃ 2min,共34cycles;72℃ 10min。
注:基因构建的片段1用引物Preu3-ΔCMeT-1F、Preu3-ΔCMeT-1R;基因构建的片段2用引物Preu3-ΔCMeT-2F、Preu3-ΔCMeT-2R;基因构建的片段3用引物Preu3-ΔCMeT-3F、Preu3-ΔCMeT-3R;基因构建的片段4用引物Preu3-ΔCMeT-4F、Preu3-ΔCMeT-4R。
其中,先将聚酮合酶Preu3分为三个片段:片段1(2.5-kb,A1-C2492)、片段2(2.6-kb,G2466-C5019)以及片段三(2.6-kb,A4993-G7572),其中CMeT结构域(碱基G5365-A6342)位于片段三内;然后将片段三内的CMeT结构域片段敲除,截分为片段3(0.4-kb,A4993-T5364)与片段4(1.3-kb,C6343-G7572)。
DNA片段回收采用Thermo Scientific GeneJET凝胶回收试剂盒(K0692),具体步骤参见其说明书。
1.2异源表达载体片段的制备:片段5(6.2-kb)通过限制性内切酶Nde I/Pme I酶切质粒YEpADH2p-FLAG-URA获得。质粒YEpADH2p-FLAG-URA为亚利桑那大学农业与生命科学学院Istvan Molnar教授馈赠。
酶切体系配置(180μL):ddH2O 132μL,10×FastDigest Green Buffer 18μL,质粒YEpADH2p-FLAG-URA18μL,限制性内切酶①(Nde I)6μL,限制性内切酶②(Pme I)6μL。37℃酶切2h后,采用Thermo Scientific GeneJET凝胶回收试剂盒(K0692)进行切胶回收,具体步骤参见其说明书。
1.3异源表达载体的构建与转化:采用SE无缝克隆和组装试剂盒(庄盟生物,ZC231)将1.1制备的preu3-ΔCMeT基因片段1-4与1.2制备的载体片段5进行重组构建异源表达载体YEpADH2p-URA-Preu3-ΔCMeT。
重组反应体系配置(10μL):5×SE Cloning Buffer 2μL;片段1 2μL;片段2 2μL;片段3 1μL;片段4 1μL;片段5 1μL;SE Recombinase 1μL。37℃反应0.5h,冰上2min,然后转化T1 Phage Resistant感受态细胞(庄盟生物,ZC102),具体步骤参见其说明书。
1.4异源表达质粒的提取及验证:大肠杆菌质粒的提取采用AxyPrepPlasmid Miniprep Kit(AP-MN-P-250),具体步骤参见其说明书。对构建的异源表达载体进行Nde I单酶切验证(7623/4773/460-bp)并测序,确证载体序列的正确性(载体酶切验证见图3)。
酶切验证体系配置(10μL):ddH2O 5.5μL,10×FastDigest Green Buffer 1μL,异源表达质粒3μL,限制性内切酶(Nde I)0.5μL。37℃酶切反应0.5h后跑胶。
2.聚酮合酶Preu3-ΔCMeT酿酒酵母异源表达突变株的构建及其代谢产物分析鉴定
2.1聚酮合酶Preu3-ΔCMeT酿酒酵母异源表达突变株的构建:采用LiAc/PEG4000介导的转化法将载体YEpADH2p-URA-Preu3-ΔCMeT转化酿酒酵母。
将制备的100μL酵母感受态细胞3600r/min,离心5min,弃上清,然后依次向管中加入78μL ddH2O,36μL LiAc(1mol/L),240μL PEG4000溶液(50%(w/v)),3μL异源表达载体(YEpADH2p-URA-Preu3-ΔCMeT),将混合液混合均匀;30℃放置30min,然后转移至42℃放置30min;将转化液3600r/min,离心5min,弃上清,用200μL无菌水重悬菌体,然后均匀涂布于SC-Ura固体平板上,30℃培养箱中培养2-4d。待酵母转化菌株长出后,将其进行发酵及代谢产物分析。
SC-Ura固体平板的配置:YNB 6.7g/L,葡萄糖20g/L,0.77g/L-ura Do Supplement(美国Clontech公司),15g琼脂,121℃灭菌15min。
2.2酵母突变株的小量发酵:在转化的平板上随机挑取两个克隆,在SC-Ura固体平板上划线,平板置于30℃培养箱过夜培养。取适量菌体(优选为接种1mL OD600nm=0.5的菌液)接种于25mL SC-Ura液体培养基中(用125mL三角瓶),30℃,220r/min,培养1d;第2d,加入25mL 1%YPD培养基,继续培养3d后进行次级代谢产物分析。
SC-Ura液体培养基的配置:YNB 6.7g/L,葡萄糖20g/L,0.77g/L-ura DoSupplement(美国Clontech公司),121℃灭菌15min。
1%YPD培养基的配置:酵母提取物10g/L,蛋白胨20g/L,葡萄糖10g/L,121℃灭菌15min。
2.3酵母突变株代谢产物分析(色谱图见图4):用等体积的乙酸乙酯萃取酿酒酵母发酵液,取乙酸乙酯相装入圆底烧瓶中旋蒸。待溶剂悬干后,加入800μL甲醇溶解代谢产物,进行LC-MS分析。色谱条件是:Reverse-phase C18 column(Kromasil 100-5-C18,4.6×250mm,5μm);流动相是水(A)/甲醇(B,加体积百分数为0.1%冰醋酸);流速0.8mL/min;进样体积5μL;DAD检测器,检测波长为300nm;柱温25℃;采用梯度洗脱,程序是起始流动相比例5%B保持5min,30min内B从5%线性升至100%,保持100%B 10min,然后1min内B从100%线性降至5%,并保持5%B 4min。质谱条件是:Q Exactive检测器;电喷雾离子(ESI)源;正、负离子切换采集;一级质谱全扫描(150-1000m/z);喷雾电压3.8kv;离子传输管温度325℃;鞘气流速40arb;辅助气流速20arb;辅助气加热温度350℃。
2.4酵母突变株目标代谢产物的分离纯化:将可产生目标代谢产物的酵母突变株大规模发酵2L,发酵及萃取方法分别同2.2,2.3,制得粗浸膏1.2g。将粗浸膏用少量(约1mL,粗浸膏可溶解的量即可)甲醇溶解后,通过ODS C18反相硅胶柱层析法,选用甲醇-水(MeOH-H2O)对粗浸膏进行梯度洗脱(流动相梯度配比(甲醇:水的体积比)依次分别为5:95、10:90、15:85、20:80、25:75,各梯度洗脱所用的流动相均为100mL),共得到五个组分段(Fr.A-E),其中目标代谢物集中在Fr.E组分中,将其通过高效液相制备(色谱条件同2.3,tR=24.1min)得到目标单体化合物约0.2g。
2.5酵母突变株目标代谢产物的结构解析:目标代谢产物为白色晶体,最大紫外吸收波长为300nm(图5),其结构经高分辨质谱(HRMS)及一维核磁共振波谱(1H-NMR、13C-NMR)鉴定,HRMS测得其分子离子峰m/z为167.03467[M-H]-以及169.04955[M+H]+(图6),分子式为C8H8O4,其结构解析所需的核磁数据见图7-图8以及表2,最终解析其结构为苔色酸(分子结构见图1)。
表2:化合物苔色酸的1H与13C核磁数据(500MHz,CD3OD,δin ppm,J in Hz)
3.苔色酸抗菌活性实验
3.1苔色酸抗农作物病原真菌活性实验:将苔色酸以及酮康唑(阳性对照)用DMSO配制成1mg/mL的母液。
将目标农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)在PDB(马铃薯葡糖糖肉汤)培养基中活化2-3d,活化好后取1mL菌液加入到100mL PDB培养基中得到稀释菌液。以酮康唑为阳性对照,取2μL目标化合物加入到198μL的目标稀释菌液里面,采用二倍稀释法测试苔色酸对以上七种农作物病原真菌的拮抗活性(活性结果见表3)。
PDB培养基配制:取23g PDB粉末(北京奥博星生物技术有限责任公司),加入1000mL蒸馏水,121℃高压灭菌15min。
3.2苔色酸抗临床耐药细菌活性实验:将苔色酸以及环丙沙星(阳性对照)用DMSO配制成1mg/mL的浓度。
将目标临床耐药细菌(耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌)在LB(溶菌肉汤)培养基中活化8h,活化好后取50μL菌液加入到50mL LB培养基中得到稀释菌液。以环丙沙星为阳性对照,取2μL目标化合物加入到198μL的目标稀释菌液里面,采用二倍稀释法测试苔色酸对以上八种临床耐药细菌的拮抗活性(活性结果见表4)。
LB培养基配制:取25g LB粉末(北京酷来搏公司),加入1000mL蒸馏水,121℃高压灭菌20min。
表3:苔色酸对七种农作物病原真菌的抑制活性(以酮康唑为阳性对照)
表4:苔色酸对八种临床耐药细菌的抑制活性(以环丙沙星为阳性对照)
4.结论
实验结果表明,我们首次以光黑壳属真菌来源的聚酮合酶Preu3(合成3-甲基苔色酸)为材料,基于组合生物合成技术构建了新型“非天然”聚酮合酶Preu3-ΔCMeT,将其转入酿酒酵母,成功获得可高效生产苔色酸的突变株(~0.1g/L);并且制备的苔色酸对耐碳青霉烯铜绿假单胞菌一种临床耐药细菌以及苹果轮纹病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌四种农作物病原真菌均有不同程度的抑制作用,其中对耐碳青霉烯铜绿假单胞菌有很强的拮抗作用(MIC,12.5μg/mL)。
苔色酸及其衍生物具有抗炎、抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性,故其可作为食品与医药行业天然抗菌剂、抗氧化剂及新型药物等的潜在生物资源。但目前,市售苔色酸(CAS:480-64-8)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高,故导致其在食品、医药等领域应用受限。在本发明中,我们首次以光黑壳属真菌来源的聚酮合酶Preu3(合成3-甲基苔色酸)为材料,基于组合生物合成技术敲除其CMeT结构域构建了新型“非天然”聚酮合酶Preu3-ΔCMeT,将其转入酿酒酵母,成功获得可高效生产苔色酸的突变株;并以制备的苔色酸为对象,研究了其对七种农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)及八种临床耐药细菌(耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌)的抑制活性。研究发现,苔色酸对耐碳青霉烯铜绿假单胞菌一种临床耐药细菌以及苹果轮纹病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌四种农作物病原真菌均有不同程度的抑制作用,其中对耐碳青霉烯铜绿假单胞菌有很强的拮抗作用(MIC,12.5μg/mL)。本发明极大地丰富了苔色酸的生产来源,对于拓展其衍生化途径,研发新型抗菌剂有重要的科学价值和应用前景。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 中南民族大学
<120> 聚酮合酶Preu3-∆CMeT及其在制备苔色酸中的应用
<160> 10
<170> SIPOSequenceListing 1.0
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Met Asn Pro Pro Ser Ala Leu Ala Phe Gly Pro Glu Glu Arg Ile Pro
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Thr Ala Ser Asn Leu Arg Leu Leu Lys Asp Val Leu Gln Asp Asp Pro
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Thr Phe Ala Gly Ile Thr Ala Cys Leu Lys Gln Leu Pro Asp Thr Trp
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Lys Ala Leu Leu His Gln Asp Ala Gln Leu Gln Ser Leu Ala Ser Glu
50 55 60
Arg Arg Ala Ala Val Leu Ser Asn Ser Leu Leu Asn Asp Glu Gln His
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Glu Gly Asp Met Asp Thr Asn Gln Val Ile Met Pro Met Thr Val Leu
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Val His Met Val Gln Tyr Arg Gln Phe Leu Gln Gln Ser Ser Pro Ser
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Ser His Ala Thr Val Met Gln Ser Val Ala Ala Gly Gly Val Gln Gly
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Phe Cys Ala Gly Leu Leu Ser Ala Phe Ala Val Cys Ser Met Thr Asn
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Glu Asp Asp Phe Asp Ala Cys Ala Thr Tyr Ala Ile Lys Leu Ala Met
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Cys Val Gly Ala Tyr Val Asp Leu Ala Met Glu Ser Glu Lys Gly Asp
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Met Ala Ser Ala Ile Val Arg Trp Ser Ile Pro Asp Gly Arg Asn Arg
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Val Asn Lys Ala Val Gly Arg Tyr Gln Ser Ala Tyr Ile Ser Ala Ile
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Ser Asp Glu Asp Asn Val Thr Val Thr Ala Ser Arg Pro Asp Leu Asp
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Ala Ile Cys Thr Ser Leu Gly Ser Thr Gly Met Ser Ser Lys Ile Leu
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Ala Met Thr Gly Ser Phe His His Pro Lys Asn Phe Asp Leu Leu Gln
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Arg Met Ile Ser Leu Leu Arg Ala Pro Gln Leu Ala Pro Ser Thr Lys
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Phe Thr Asn Ala Leu Leu Arg Ser Asn Ser Thr Gly Glu Leu Leu Thr
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Gly Ala Lys Thr Ile Glu Asn Ile Leu Glu Asp Ile Leu Cys Lys Thr
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Ala Asp Trp Arg Leu Thr Met Ala Asn Thr Ser Lys Ala Leu Arg Ser
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Gly Asn Gly Ser Arg Pro Asn Ile His Thr Phe Gly Leu Val Glu Phe
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Ile Pro Ser Phe Val Lys Asn Glu Phe Asn Ile Leu Thr Gln Arg Leu
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Ala Pro Thr Ala Lys Glu Gln Thr Gly Ala Ser Pro Ser Lys Ser Thr
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Leu Gln Tyr Asn Asp Asn Ala Val Ala Val Val Gly Met Ala Cys Arg
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Phe Pro Gly Ala Asp Asp Leu Asp Glu Phe Trp Glu Leu Leu Gln Ser
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Gly Lys Ser Met His Glu Arg Met Pro Ala Asp Arg Phe Ser Thr Thr
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Gly Leu Arg Arg Ser Asn Asp Gly Ala Pro Phe Trp Gly Asn Phe Leu
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Lys Asp Ile Asp Ala Phe Asp His Gln Phe Phe Lys Lys Ser Ser Arg
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Glu Ala Ala Ala Met Asp Pro Gln Gln Arg Leu Leu Leu Gln Cys Ala
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Tyr Val Ala Met Glu Asn Ala Gly Tyr Phe Asp Pro Ser Val Gln His
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Lys Ile Arg Asp Thr Gly Val Tyr Leu Gly Ala Cys Ser Ser Asp Tyr
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Asn Asp Asn Val Ala Ser His Lys Pro Thr Ala Tyr Ser Thr Leu Gly
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Thr Leu Arg Ala Phe Leu Thr Gly Arg Ile Ser His Tyr Phe Asp Trp
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Thr Gly Pro Ser Val Val Tyr Asp Thr Ala Cys Ser Ser Ser Ala Val
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Ala Ile Asp Ala Ala Cys Lys Ala Ile Leu Ala Gly Asp Cys Gln Gln
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Ala Leu Ala Gly Gly Val Ser Leu Tyr Thr Ser Pro Asn Phe Tyr Gln
565 570 575
Asn Leu Asp Ala Ala Ser Phe Leu Ser Gln Thr Gly Pro Cys Lys Pro
580 585 590
Phe Asp Ala Asn Ala Asp Gly Tyr Cys Arg Gly Glu Gly Val Gly Leu
595 600 605
Val Val Leu Lys Lys Leu Ser Asp Ala Ile Arg Cys Gly Asp Lys Ile
610 615 620
Val Ala Val Ile Ala Ser Thr Gly Val Asn Gln Asn Arg Asn Cys Thr
625 630 635 640
Gly Ile Thr Val Pro His Gly Gly Ser Gln Ala Asp Leu Tyr Arg Arg
645 650 655
Val Val Ala Lys Ser Gly Leu Asn Ala Ser Gln Val Ser Tyr Val Glu
660 665 670
Ala His Gly Thr Gly Thr Pro Val Gly Asp Pro Ile Glu Phe Thr Ser
675 680 685
Ile Lys Ser Val Phe Ala Asn Pro Asp Ile Thr Arg Asp Glu Pro Leu
690 695 700
Thr Ile Ala Ser Val Lys Gly Ser Ile Gly His Leu Glu Gly Ala Ala
705 710 715 720
Gly Val Ala Ser Leu Ile Lys Val Cys Leu Met Leu Gln His Ser Ala
725 730 735
Ile Pro Pro Gln Ala Asn Phe Thr Lys Pro Asn Pro Asn Leu Gly Gly
740 745 750
Val Asp Met Arg Asn Ile Val Ile Pro Thr Ser Ser Ile Pro Trp Lys
755 760 765
Ala Arg Asn Lys Val Ala Cys Ile Asn Asn Tyr Gly Ala Ala Gly Ser
770 775 780
Asn Gly Ala Met Ile Val Cys Gln Pro Ser Glu Pro Ala Ser Lys Thr
785 790 795 800
Gln Thr Arg Leu Pro Ser Gln Ser Leu Ser Tyr Pro Leu Phe Ile Ser
805 810 815
Gly Asp Gly Thr Asp Ala Val Glu Ala Asn Cys Arg Ala Ile Ala Lys
820 825 830
Tyr Ala Arg Gln Leu Gln Gln Lys Arg Ala Pro Ser Val Val Ala Ser
835 840 845
Leu Ala Tyr Arg Leu Ala Thr Ser Gln Asn Gln Asn Leu Ser Tyr Ala
850 855 860
Met Val Thr Thr Ile Ser Glu Asn Gly Asp Ile Glu Ser Thr Leu Thr
865 870 875 880
Lys Ala Ser Ala Thr Leu Thr Gln Pro Arg Ser Lys Ala Lys Gln Ser
885 890 895
Val Val Leu Cys Phe Gly Gly Gln Val Lys Ala Phe Val Gly Leu Asp
900 905 910
Gln Gln Leu Phe Asp Ser Ser Ser Ile Leu Gln Lys His Leu Arg Leu
915 920 925
Cys Asp Ser Thr Met Arg Asp Leu Gly Tyr Pro Ser Ile Phe Pro Ala
930 935 940
Ile Phe Gln Ser Glu Pro Leu Lys Asp Pro Val Gln Leu His Gly Val
945 950 955 960
Leu Phe Ala Met Gln Tyr Ser Ser Ala Lys Ala Trp Leu Asp Cys Ser
965 970 975
Leu Gln Val Asp Ala Val Val Gly His Ser Phe Gly Gln Leu Thr Ala
980 985 990
Leu Thr Val Ala Gly Val Leu Ser Leu Lys Asp Gly Leu Arg Leu Val
995 1000 1005
Cys Gly Arg Ala His Leu Ile Lys Thr Lys Trp Gly Ser Ala Thr Gly
1010 1015 1020
Ala Met Ile Ala Ile Glu Ala Pro Leu Ile Arg Val Gln Glu Ile Leu
1025 1030 1035 1040
Ser Lys Ile Ser Val Ala Gly His Glu Ala Glu Ile Ala Cys Tyr Asn
1045 1050 1055
Ala Arg Glu Ser His Val Leu Val Gly Thr Thr Thr Ala Ile Asp Ala
1060 1065 1070
Val Arg Thr Phe Val Leu Glu Ser Gly Ile Lys His Lys Arg Leu Pro
1075 1080 1085
Val Thr His Gly Phe His Ser Thr Phe Thr Glu Ala Leu Leu Pro Gly
1090 1095 1100
Leu Arg Glu Leu Ala Lys Gly Leu Gln Phe Lys Ser Pro Ile Ile Pro
1105 1110 1115 1120
Ile Glu Thr Cys Thr Glu Tyr Lys Ser Trp Glu Gln Ala Thr Ala Asp
1125 1130 1135
Met Ile Thr Lys His Thr Arg Glu Pro Val Tyr Phe Val His Ala Ile
1140 1145 1150
Glu Arg Leu Ser Ala Arg Leu Gly Pro Cys Thr Trp Val Glu Ala Gly
1155 1160 1165
Thr Gly Ala Ser Thr Pro Ala Met Ile Lys Arg Cys Leu Pro Asp Ser
1170 1175 1180
Cys Ala Asp Ser Phe Ile His Ala Thr Leu Glu Ser Asn Lys Ala Phe
1185 1190 1195 1200
Gly Ser Leu Ala Asp Ala Thr Ala Asn Leu Trp Arg Cys Ser Gln Pro
1205 1210 1215
Val Gln Phe Trp Pro Phe His Ala Ser Asp Arg Gly Arg Tyr Val Pro
1220 1225 1230
Leu Asn Leu Pro Gly Tyr Gln Phe Arg Lys Thr Lys His Trp Leu Glu
1235 1240 1245
Trp Gln Asp Thr Val Ala Leu Pro Ala Phe Leu Glu Lys Glu Pro Ser
1250 1255 1260
Thr Ser Glu Pro Lys Gly His Glu Leu Leu Thr Phe Ser Ser Phe Glu
1265 1270 1275 1280
Asp Thr Ser Lys Ser Val Ala Ala Phe Lys Val Asp Pro Glu Ser Asp
1285 1290 1295
Glu Phe Met Met Leu Val Lys Gly His Ala Val Val Ala Gln Pro Leu
1300 1305 1310
Cys Pro Ala Pro Leu Tyr Cys Glu Leu Ala Leu Arg Ala Ile Lys His
1315 1320 1325
Leu Ser Pro Glu Thr Ala Ser Asn Ala Pro Asp Ile Arg Asp Leu Gln
1330 1335 1340
Ile His Ala Pro Leu Gly Leu Lys Thr Asn Arg Asn Ile Arg Leu Val
1345 1350 1355 1360
Val Gln Lys Asn Ser Ile Pro Gly His Trp Thr Phe Thr Val Lys Ser
1365 1370 1375
Ser Met Gly Ser Asp Asp Glu Leu Thr His Ala His Gly Leu Val Ala
1380 1385 1390
Phe Gly Gly Thr Val Glu Gln Glu Leu Ala Ser Tyr Gln Arg Leu Ile
1395 1400 1405
Gly His Gln Lys Ile Gln Ser Leu Met Thr Asp Pro Glu Cys Asp Ala
1410 1415 1420
Leu Arg Gly Ser Ala Thr Tyr Lys Ala Phe Asn Arg Val Val Thr Tyr
1425 1430 1435 1440
Ser Ser Tyr Tyr Lys Gly Val Gln Ala Ile Tyr Gly Arg Gln Asn Glu
1445 1450 1455
Ala Cys Gly Lys Ile Glu Leu Ser Ser Gly Glu Glu Gln Met Ala Gln
1460 1465 1470
Ala Arg Gly Ile Leu Thr Pro Leu Leu Ala Asp Asn Phe Ile Gln Ile
1475 1480 1485
Ala Gly Leu Gln Ile Asn Val Leu Gly Asp Cys Glu Asp His Leu Val
1490 1495 1500
Phe Val Cys Thr Glu Thr Gln Arg Ile Ile Tyr Gly Pro Gly Leu His
1505 1510 1515 1520
Gln Gln Pro Ala Ala Arg Tyr Glu Val Tyr Ser Thr Ile Ser Gln Asn
1525 1530 1535
Gly Pro Lys Glu Val Met Ser Asp Val Val Val Phe Asp Pro Ala Thr
1540 1545 1550
Lys Asn Val Glu Phe Val Ala Leu Gly Cys Arg Phe Thr Arg Val Thr
1555 1560 1565
Val Pro Gly Leu Arg Asn Ala Leu Gln Ala Ala Asn Gly Asp Ala Arg
1570 1575 1580
Ala Gln Glu Arg Pro Ser Gly Ser Arg Ile Ser Pro Ser Pro Leu Ala
1585 1590 1595 1600
Pro Glu Leu Pro Ala Lys Ile Gln Ile Gln Ser Arg Glu Asn Leu Asp
1605 1610 1615
Ile Thr Glu Lys Ser Gly Arg Gly Lys Pro Pro Arg Val Glu Asn Ile
1620 1625 1630
Gln Ile Ala Thr Pro Lys Val Asp Tyr Leu Ala Gln Val Lys Ala Leu
1635 1640 1645
Leu His Lys Val Ser Asp Val Pro Ile Asp Thr Ile Gln Lys Asp Ser
1650 1655 1660
Thr Leu Asp Asp Leu Gly Ile Asp Ser Leu Met Val Met Glu Val Gln
1665 1670 1675 1680
Thr Glu Val His Ser Glu Phe Gln Leu Thr Ile Pro Asn Lys Asp Trp
1685 1690 1695
Ala Thr Leu Glu Thr Pro Gly Lys Leu Ala Glu Tyr Leu Ala Lys Thr
1700 1705 1710
Leu Gly Gly Ser Val Pro Asp Ser Ala Pro Pro Gly Val Gln Arg Val
1715 1720 1725
Pro Ala Leu Val Ile Ser Asp Ala Glu Gln Ser Ser Asp Glu Ser Pro
1730 1735 1740
Tyr Asp Ser Thr Asp Asp Ser Ala Ser Gly Tyr Gly Asp Leu Asp Ile
1745 1750 1755 1760
Asp Thr Ala Ala Thr Thr Pro Gly Ile Phe Ala Thr Arg Asp Ser Ser
1765 1770 1775
Pro Phe Arg Lys Ala Ala Leu Asp Ser Pro Asn Pro Arg Pro His Val
1780 1785 1790
Leu Ala Asp Glu Asn Leu Trp Asp Gln Ser Leu Arg Ala Ala Gly Tyr
1795 1800 1805
Gly Asp Val Gln Trp Thr Glu Gly Gln Ser Glu Glu Ser Lys Thr Leu
1810 1815 1820
Arg Leu Ile Ala Ala Phe Asn Val Ser Asn Glu Asp Ala Lys Ala Ala
1825 1830 1835 1840
Asn Ala Leu Ala Ser Ala Leu Ala Val Pro Gly Arg Lys Gly Arg Thr
1845 1850 1855
Ser Ala Thr Thr Ile Arg Trp Lys Gln Glu Gly Asp Leu Asp Leu Met
1860 1865 1870
Ala Asp Val Tyr Leu Pro Ser Asp Leu Asp Ala Ser Thr Val Ser Arg
1875 1880 1885
Pro Val Ala Leu Ile Leu His Gly Gly Gly His Val Leu His Thr Arg
1890 1895 1900
Lys His Ile Asn Pro Arg His Ile Lys Met Leu Gln Asp Leu Gly Phe
1905 1910 1915 1920
Leu Pro Val Ser Val Asp Tyr Arg Leu Cys Pro Glu Val Asn Ile Arg
1925 1930 1935
Asp Gly Pro Met Thr Asp Ala Cys Glu Ala Val Asp Trp Ala Arg Asn
1940 1945 1950
Ile Leu Pro Cys Leu Pro Val Cys Ser Glu Leu Arg Val Asp Lys Glu
1955 1960 1965
His Val Val Val Ile Gly Tyr Ser Thr Gly Gly His Leu Ala Leu Thr
1970 1975 1980
Thr Ala Phe Thr Thr Arg Val Arg Gly Phe Lys Pro Pro Ser Ala Ile
1985 1990 1995 2000
Leu Gly Phe Tyr Cys Pro Thr Asn Tyr Ser Ala Asp Trp Trp Arg Ser
2005 2010 2015
Pro Ile Tyr Pro Glu Leu Ala Gln Gln Ser Ser Ser Glu Thr Phe Asp
2020 2025 2030
Leu Leu Glu Gly Val Asn Glu His Ala Ile Ala Gly Tyr Thr Pro Thr
2035 2040 2045
Val Asn Asn Asn Val Ala Ala Leu Leu Met Ser Leu Asp Asp Pro Arg
2050 2055 2060
Trp Arg Phe Val Leu His Ala Asn Trp Arg Ala Gln Thr Leu Pro Met
2065 2070 2075 2080
Leu Ile Asn Gly Leu Pro Ser Lys Ser Arg Leu Ala Arg Ser Gly Gln
2085 2090 2095
Thr Val Asp Ser Val Ile Asn Arg Glu Ile Pro Asp Ala Glu Asp Val
2100 2105 2110
Ala Ser Ile Ser Pro Tyr Asp Gln Ile Val Arg Gly Ser Tyr Ser Thr
2115 2120 2125
Pro Thr Phe Leu Leu His Gly Thr Lys Asp Asp Leu Ile Pro Trp Gln
2130 2135 2140
Gln Ser Ile Ala Thr Val Asp Ala Leu Ala Arg Arg Gly Val Asn Ala
2145 2150 2155 2160
Arg Val Glu Ile Ile Glu Gly Ala Glu His Cys Phe Asp Val Trp Ser
2165 2170 2175
Asp Lys Tyr Asp Gly Met Ile Gly Arg Ala Leu Glu Trp Leu Val Glu
2180 2185 2190
Gln Cys Arg Asn Ala
2195
<210> 2
<211> 6594
<212> DNA/RNA
<213> 人工序列()
<400> 2
atgaaccctc ctagcgcttt ggcctttggt cccgaagagc gcatcccaac tgcttcaaac 60
ttgcgacttc taaaggatgt tttgcaagac gatccgacat tcgccggcat tactgcctgc 120
ctgaagcagt taccagatac ctggaaagcc ttgctccatc aagatgcaca attacaatcg 180
cttgcaagcg agaggcgtgc tgctgttctg tcgaactcgc ttctcaatga cgagcagcat 240
gaaggtgaca tggacaccaa ccaggtcatc atgcccatga cagtcctcgt ccacatggtg 300
caataccgtc agttcttgca gcagagctct ccttccagcc atgctacagt catgcagagc 360
gtcgcagccg gaggtgtgca aggcttctgt gcgggtcttc tttccgcctt tgctgtgtgt 420
tcgatgacca acgaggacga ctttgacgca tgtgctacat atgcgatcaa gctagccatg 480
tgcgtgggtg cctatgttga cctggccatg gagtcagaga agggggacat ggcatcagct 540
attgttcgct ggtcaatacc cgatggtcga aaccgtgtca acaaagccgt tggacgctat 600
cagagtgctt atatttctgc tatatctgac gaggacaacg ttacagtaac agccagtcga 660
cccgatctcg atgcgatttg cacctccctt ggaagcacag gcatgtccag caagattttg 720
gccatgactg ggagtttcca ccaccccaag aattttgacc tgctccaacg tatgatatcg 780
ctccttaggg ctccacagct cgccccgtcc acgaaattca ctaatgcgct gctccgctcc 840
aatagtactg gggaattact cactggtgcg aagacaattg agaatatcct agaagacatt 900
ctctgcaaga ctgctgattg gcggttgact atggccaaca cctcaaaagc gttgagatca 960
ggaaacggca gccgacccaa cattcataca ttcgggttgg tcgaattcat accttccttc 1020
gtaaaaaatg aattcaacat ccttacacag cggttggcgc cgaccgcgaa agagcagact 1080
ggagcttctc caagcaagtc tactctccag tacaacgata atgcagtcgc cgtggtcgga 1140
atggcctgcc gtttccccgg tgcagatgac ttggatgagt tttgggaact ccttcagtcc 1200
gggaagtcga tgcacgagcg tatgccagca gatcgtttca gcactacagg tctccgacgg 1260
tccaacgatg gtgctccgtt ttggggtaac ttcctcaaag atatcgacgc cttcgatcac 1320
caattcttca agaagtctag ccgcgaggca gctgcgatgg atccccaaca gcgtcttctc 1380
ctccagtgcg cttacgtggc catggaaaac gcgggatact ttgatccatc tgttcagcac 1440
aagatcagag acactggagt ttacctcgga gcatgctcta gcgactacaa tgacaatgtg 1500
gctagccata agcccacagc ttattccacg ctggggacgc tcagggcgtt tctgactggt 1560
cgtatcagtc actactttga ttggaccgga ccctccgttg tctacgacac agcttgttcg 1620
tcgtctgccg tggctatcga cgcagcttgc aaagccatcc ttgctggtga ttgtcaacag 1680
gctttggcag gtggagtctc tttgtacaca tcgcccaact tctatcagaa cttggacgct 1740
gcttcattcc tcagccagac cggtccctgc aagccctttg atgcgaacgc tgacggctac 1800
tgtcgaggcg agggcgtggg attggtggtc ctcaagaagc tgagcgatgc tattcggtgc 1860
ggcgataaga ttgtagccgt cattgctagc acgggggtca accaaaaccg caattgcacg 1920
ggcatcaccg ttcctcatgg tgggtcgcaa gctgatcttt accggagagt tgtagccaag 1980
agtggcttga atgcctcaca ggtttcctat gtggaagcgc atggaacggg cacccccgtc 2040
ggcgatccaa tcgagtttac gagcatcaaa tctgtcttcg caaatcctga cataacccga 2100
gacgagccgt tgactattgc atcggtgaag ggcagcatcg ggcatctcga aggcgcagca 2160
ggagttgcat cgctgatcaa ggtttgcctg atgctacagc actctgctat tcctccccag 2220
gcaaacttta cgaagcctaa ccccaacctt gggggggtcg atatgcgcaa catcgtcata 2280
ccaaccagca gcatcccgtg gaaggcgagg aacaaggttg cgtgcatcaa caactatggt 2340
gcagctggca gcaatggcgc catgattgta tgccagcctt ccgagccagc atcaaagaca 2400
caaacgagac tcccatcgca aagcctttcg tatcctctgt ttatatccgg agatggcact 2460
gatgcggtgg aagccaattg tcgggccatt gccaaatatg ctcgccaact ccagcaaaag 2520
cgagcaccgt ctgtggtggc cagtctcgcg tacaggttgg caacgtccca gaatcaaaat 2580
ctgtcgtatg caatggtgac tacaatctct gaaaatgggg atatcgaaag cacattgacc 2640
aaagcgtccg ctaccttgac gcagcctcgc tcgaaggcaa aacaatcagt tgtgctgtgt 2700
tttggcggcc aagtaaaagc ttttgtgggt ctggaccagc agctgtttga ttcctccagt 2760
attcttcaga aacatctgcg gctctgcgac tcgaccatgc gtgatctggg ctatcccagc 2820
atattcccag ccatcttcca gtcggagcct ctgaaggacc cagtacagct ccatggtgta 2880
ctattcgcga tgcagtattc atctgcgaaa gcttggctgg attgcagtct ccaggttgat 2940
gctgtcgttg gtcacagttt tggacagctt actgctctta ctgttgccgg cgtccttagt 3000
ctgaaggatg gcttgaggct tgtatgcggc cgggcacacc taattaagac gaagtggggc 3060
tcagccactg gggcaatgat tgcaatcgaa gcgcctctaa tccgtgtgca ggaaatcctc 3120
tcgaaaatct ccgtcgccgg acatgaagca gagatcgcgt gctacaatgc tcgagagagc 3180
catgtcctgg tcggtaccac tactgcgatt gatgctgtcc ggacattcgt acttgagagt 3240
ggtatcaagc ataagcgttt gccggtaacc catggatttc actctacctt cacggaagcc 3300
ctgttacctg gtctccgtga gctggcgaaa ggtctacagt tcaagtcccc aatcattccg 3360
atagagacat gcacagaata caagagctgg gagcaggcta cagctgacat gatcacgaaa 3420
cacacgaggg agcctgttta cttcgtccat gcgatagaaa gactctcggc tcggttgggg 3480
ccatgcacat gggttgaggc aggtaccggc gcatcgactc ctgcaatgat caagagatgc 3540
cttccggatt cttgtgcaga cagcttcata cacgcgaccc ttgaatccaa caaagctttc 3600
ggatcgctag ccgatgccac tgcgaatcta tggcgatgca gccagcccgt ccagttctgg 3660
cctttccatg caagcgacag agggagatat gtcccactta acttgccggg ctaccaattc 3720
cgtaagacga agcattggct cgagtggcaa gacaccgtcg ctttgcctgc ttttctagaa 3780
aaagaaccat cgacctctga gcctaaaggg catgagctgc taaccttctc ttcgttcgag 3840
gacacttcaa aatctgttgc agcattcaaa gtggaccctg aaagcgatga attcatgatg 3900
ctggtcaaag gtcatgctgt ggttgctcag ccgctttgcc cggcccccct gtactgcgag 3960
cttgccctac gagctatcaa gcatctctcg ccggaaactg cgtcaaatgc cccagatatc 4020
cgtgatctgc agatccatgc tccccttgga ctgaaaacta atcgaaacat ccgccttgta 4080
gtccagaaga acagtatccc aggtcactgg actttcaccg tgaagagctc aatgggatcc 4140
gacgacgaac ttactcacgc tcatgggctt gtagcattcg gtggaactgt ggaacaggag 4200
ttggcatcgt atcagcgact tatcggtcac cagaagatac agtctctgat gacagacccg 4260
gaatgtgatg ctttgagggg ctccgcgaca tacaaagcgt tcaaccgagt cgtcacatat 4320
tcttcctact acaaaggtgt ccaagcgata tatggtcgac agaacgaagc ttgtgggaag 4380
attgagttgt catcaggcga agaacaaatg gcccaggcac gcggaatact gactcccctc 4440
cttgccgata atttcataca gattgccggt ttgcagatca atgtgctggg agattgcgag 4500
gatcacttgg tcttcgtgtg tacggaaaca caacggatca tttatgggcc aggactccat 4560
caacaaccgg ctgctcgata tgaggtctat tcgacgattt ctcagaacgg ccccaaggaa 4620
gtcatgagtg acgttgtcgt tttcgatccc gccaccaaga atgttgagtt cgtcgcactg 4680
ggttgccgct tcacgcgagt gacagtacca ggccttcgca acgcacttca agccgccaat 4740
ggagacgctc gagctcagga gcggcctagc ggatctcgca tcagccccag tccgcttgca 4800
cctgaattgc ccgcgaagat tcaaattcag tcacgtgaga atctggacat cacggaaaag 4860
tctggacgtg gaaagcctcc tcgtgttgag aatattcaga tcgccacacc aaaggtcgac 4920
tacttggctc aggtgaaggc gcttctccac aaagtttccg atgtccccat cgataccata 4980
caaaaggatt cgacgcttga cgatctgggc attgattcgc ttatggtcat ggaggtgcaa 5040
actgaggtac actccgaatt ccaactgacc atacccaaca aagactgggc tacgttggag 5100
actcctggga agctcgccga atatcttgca aagacgctag ggggttctgt ccctgatagc 5160
gcacctccag gtgtacaacg tgtccctgcg ttagtaatct cggatgctga acagagttcc 5220
gacgaaagcc catatgacag cacagatgat tcagctagcg gttacggtga tctagacatc 5280
gatactgccg cgaccacgcc tggaatcttc gccactcgtg atagttcgcc attccggaaa 5340
gccgcgttgg actcccctaa ccctcgacca cacgttcttg ctgacgaaaa tctctgggac 5400
cagagcttac gagcagctgg ttatggagac gtgcagtgga ctgaagggca gtctgaggaa 5460
tccaagacgc tacgactcat tgctgcgttc aacgtaagca acgaggatgc gaaggcagcg 5520
aacgctctgg cgagcgcctt ggccgtaccc gggcgtaaag gcaggaccag tgcgacgacc 5580
atacgctgga agcaagaggg cgatctggat ctcatggcgg atgtatacct accgtctgat 5640
ctggatgcat caactgtaag cagacccgtc gctctgatcc ttcacggtgg cggccatgtc 5700
ttgcacacta gaaaacacat caacccgcga cacatcaaaa tgctacaaga tcttggcttc 5760
ttaccggttt cagtcgatta tcgtctttgt ccagaggtca acattcgcga tggacctatg 5820
acggacgcat gtgaggcggt ggactgggca aggaatattc taccatgtct gcctgtgtgc 5880
tccgagttgc gtgtcgacaa agagcacgta gtggtgattg gctactctac aggcggacac 5940
cttgctctga caactgcatt cacaacacgt gtcaggggat tcaagccccc ctctgccatt 6000
ctgggattct actgcccgac caactatagc gccgactggt ggcggtctcc catctatccg 6060
gagcttgctc agcagtctag ctcggagact ttcgacttgc tggaaggtgt caatgagcat 6120
gcgattgctg gatacacacc aacagtgaat aataatgtcg ctgcgcttct gatgtctctt 6180
gacgacccac gctggcgctt cgtcctacat gccaactggc gagctcagac tctcccgatg 6240
cttatcaacg ggctgccctc taagtctcgg cttgcgcgca gtggacaaac ggtggacagt 6300
gtcatcaaca gggagattcc agacgctgaa gatgttgcgt ctatcagccc gtatgatcaa 6360
atcgtcagag gaagctacag tacccctaca ttccttctcc atggaacgaa ggacgatctc 6420
attccttggc agcagagcat agcgacagtt gatgctctgg cacgacgtgg ggtgaatgcg 6480
agagtggaga ttattgaggg tgcggagcac tgttttgatg tttggtctga taagtatgat 6540
gggatgattg ggagggcact ggagtggttg gtggagcagt gtcggaatgc ttag 6594
<210> 3
<211> 59
<212> DNA
<213> 人工序列()
<400> 3
gactacaaag acgatgacga caagcttcat atgaaccctc ctagcgcttt ggcctttgg 59
<210> 4
<211> 54
<212> DNA
<213> 人工序列()
<400> 4
gcaatggccc gacaattggc ttccaccgca tcagtgccat ctccggatat aaac 54
<210> 5
<211> 32
<212> DNA
<213> 人工序列()
<400> 5
ggtggaagcc aattgtcggg ccattgccaa at 32
<210> 6
<211> 27
<212> DNA
<213> 人工序列()
<400> 6
cgaatcaatg cccagatcgt caagcgt 27
<210> 7
<211> 54
<212> DNA
<213> 人工序列()
<400> 7
acgcttgacg atctgggcat tgattcgctt atggtcatgg aggtgcaaac tgag 54
<210> 8
<211> 27
<212> DNA
<213> 人工序列()
<400> 8
agggttaggg gagtccaacg cggcttt 27
<210> 9
<211> 57
<212> DNA
<213> 人工序列()
<400> 9
cggaaagccg cgttggactc ccctaaccct cgaccacacg ttcttgctga cgaaaat 57
<210> 10
<211> 58
<212> DNA
<213> 人工序列()
<400> 10
agtgatggtg atggtgatgt ccgtttaaac ctaagcattc cgacactgct ccaccaac 58
Claims (10)
1.聚酮合酶Preu3-△CMeT,其特征在于,所述聚酮合酶Preu3-聚酮合酶Preu3-△CMeT及其在制备苔色酸中的应用CMeT的氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述聚酮合酶Preu3-△CMeT的基因。
3.根据权利要求2所述的基因,其特征在于,所述基因的编码序列是SEQ ID NO.2所示的核苷酸序列。
4.含有权利要求2或3所示的基因的重组载体。
5.一种用权利要求4所述重组载体转化得到的重组基因工程菌。
6.权利要求1所述的聚酮合酶Preu3-△CMeT在制备苔色酸中的应用。
7.一株可高效生产苔色酸的酵母突变株的构建方法,其特征在于,包括如下步骤:
采用LiAc/PEG4000介导的转化法利用权利要求4所述的重组载体转化酿酒酵母,将转化液均匀涂布于SC-Ura固体平板上,30℃培养箱中培养,即得到酵母突变株。
8.苔色酸的制备方法,采用权利要求7所述的构建方法构建的酵母突变株,其特征在于,包括如下步骤:
步骤(1),将酵母突变株接种于SC-Ura液体培养基中,30℃,摇床培养;培养的第2d,加入1%YPD培养基继续培养3-4d,得到发酵液;
其中,1%YPD培养基和SC-Ura液体培养基体积相同;
步骤(2),对步骤(1)培养得到的发酵液,用等体积的乙酸乙酯萃取,对萃取液浓缩,得到粗浸膏;
步骤(3),对步骤(2)得到的粗浸膏提纯,得到苔色酸。
9.根据权利要求8所述的苔色酸的制备方法,其特征在于:
步骤(3)中,对步骤(2)得到的粗浸膏进行提纯,提纯方法为:将粗浸膏用甲醇溶解后,通过ODS C18反相硅胶柱层析法,选用甲醇-水为流动相对粗浸膏进行梯度洗脱,共得到五个组分段Fr.A-Fr.E;目标代谢物苔色酸集中在Fr.E组分中,将Fr.E组分通过高效液相制备得到苔色酸;
其中,梯度洗脱流动相采用的甲醇:水的体积比依次为5:95、10:90、15:85、20:80、25:75,各梯度洗脱所用的流动相总体积均为SC-Ura液体培养基体积的十分之一。
10.权利要求8或9所述的苔色酸的制备方法制得的苔色酸在制备抗农作物病原真菌药物或临床耐药细菌药物中的应用,其特征在于,所述的农作物病原真菌为苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌;所述的临床耐药细菌为耐甲氧西林金黄色葡萄球菌、耐碳青霉烯大肠埃希菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯肺炎克雷伯菌、多耐药性表皮葡萄球菌。
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