CN113583993B - 聚酮合酶PreuA及其在制备红粉苔酸中的应用 - Google Patents
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Abstract
本发明涉及一种聚酮合酶及其在制备红粉苔酸中的应用,属于微生物化学技术领域。本发明首次从光黑壳属真菌中克隆到与红粉苔酸合成相关的聚酮合酶PreuA,并基于酿酒酵母异源表达技术,构建了可高效生产红粉苔酸的酵母突变株;并以制备的红粉苔酸为对象,研究了其对七种农作物病原真菌的抑制活性。研究发现,红粉苔酸对苹果轮纹病菌、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,其中对苹果轮纹病菌有很强的拮抗作用(MIC,25μg/mL)。通过本发明极大地丰富了红粉苔酸的生产来源,对于拓展其衍生化途径,研发新型生物农药有重要的科学价值和应用前景。
Description
技术领域
本发明属于微生物化学技术领域,具体涉及一种聚酮合酶及其在制备红粉苔酸中的应用。
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(Keller,2019)。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(mycophenolicacid)、肌动蛋白和血管生成抑制剂细胞松弛素E(cytochalasin E)等(Hussain et al.,2017)。PKs生物合成的关键酶——聚酮合酶(polyketide synthase,PKS)结构复杂且催化机理独特,可为研究酶催化、分子识别和蛋白质互作的分子机制提供关键工具和化学分子探针(Van Lanen&Shen,2008)。
红粉苔酸(lecanoric acid,结构见图1)为聚酮化合物苔色酸(orsellinic acid,结构见图1)的二聚体,多从天然地衣(lichen)中分离,在真菌中首次从梨孢霉属(Pyricularia sp.)中分离(Umezawa et al.,1974)。经研究报道,红粉苔酸具有抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性(Choudhary et al.,2011;Lopes et al.,2008;Luo etal.,2009;et al.,2016;Seo et al.,2009),故其可作为食品与医药行业天然抗菌剂、抗氧化剂等的潜在生物资源。但目前,市售红粉苔酸(CAS:480-56-8)均通过化学合成方式生产(Fischer E.&Fischer H.,1913;Koller&Pfeiffer,1933),售价约2000元/mg,产量低,售价高,故导致其在食品、医药等领域应用受限。因此如何克服红粉苔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红粉苔酸现有制备技术的不足,提供一种聚酮合酶及其在制备红粉苔酸中的应用。
为实现上述目的,本发明采用的技术方案如下:
本发明第一方面提供聚酮合酶PreuA,所述聚酮合酶PreuA的氨基酸序列如SEQ IDNO.1所示。
本发明第二方面提供编码权利要求1所述聚酮合酶PreuA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。
本发明第三方面提供含有上述基因的重组载体。
本发明第四方面提供所述重组载体转化得到的重组基因工程菌。
本发明第五方面提供所述的聚酮合酶PreuA在制备红粉苔酸中的应用。
本发明第六方面提供一株可高效生产红粉苔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。
本发明第七方面提供红粉苔酸的制备方法,采用上述构建方法构建的酵母突变株,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,得到红粉苔酸。
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:依次采用正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇对粗浸膏进行洗涤,回收乙酸乙酯、甲醇洗涤液,浓缩后合并,即得;其中,所使用的正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇的体积为SC-Ura液体培养基体积的十分之一。
本发明第八方面提供上述红粉苔酸的制备方法制得的红粉苔酸在制备抗农作物病原真菌药物中的应用,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。
在本发明中,我们首次从光黑壳属真菌(Preussia isomera)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA,并基于酿酒酵母异源表达技术,构建了一株可高效生产红粉苔酸的酵母突变株(红粉苔酸的产量约为0.5g/L);并以制备的红粉苔酸为对象,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)的抑制活性。研究发现,红粉苔酸对苹果轮纹病菌、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,其中对苹果轮纹病菌有很强的拮抗作用(MIC,25μg/mL)。本发明首次构建了一株可高效生产聚酮分子红粉苔酸的酵母突变株,极大地丰富了其生产来源,对于拓展其衍生化途径,研发新型生物农药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.目前市售红粉苔酸(CAS:480-56-8)均通过化学合成方式生产,售价约2000元/mg,产量低,售价高。本发明基于异源表达技术,构建了一株可高效生产红粉苔酸的酵母突变株(~0.5g/L),丰富了红粉苔酸的生产来源并可极大地提升其产量,具有可观的应用前景。
2.本发明同时也首次研究了红粉苔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)的抑制活性。研究发现,红粉苔酸对苹果轮纹病菌有很强的拮抗作用(MIC,25μg/mL),有望研发新型生物农药。
附图说明
图1为红粉苔酸(lecanoric acid)与苔色酸(orsellinic acid)的化学结构式;其中,(a)为红粉苔酸,(b)为苔色酸;
图2为聚酮合酶PreuA异源表达载体YEpADH2p-URA-PreuA的质粒图谱;
图3为聚酮合酶PreuA异源表达载体YEpADH2p-URA-PreuA的酶切验证图;其中,M,Trans 2K marker;Line 1&2,载体YEpADH2p-URA-PreuA
图4为聚酮合酶PreuA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)聚酮合酶PreuA酿酒酵母突变株;
图5为聚酮合酶PreuA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PreuA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)正离子模式;
图7为化合物红粉苔酸的1H核磁光谱图(氘代甲醇,500MHz);
图8为化合物红粉苔酸的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。
本发明中我们从光黑壳属真菌(Preussia isomera,GenBank编号为MK300824.1,菌株现保存于中南民族大学药学院杨小龙教授课题组)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA,并基于酿酒酵母异源表达技术,构建了一株可高效生产聚酮分子红粉苔酸(化合物分子结构见图1)的酵母突变株。本实验的大致步骤为:首先基于同源重组原理,构建聚酮合酶PreuA的异源表达载体YEpADH2p-URA-PreuA(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红粉苔酸(~0.5g/L);最后,以制备的红粉苔酸为对象,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)的抑制活性(实验结果见表3)。具体实验过程如下。
1.聚酮合酶PreuA异源表达载体YEpADH2p-URA-PreuA的构建
1.1聚酮合酶基因preuA的克隆:聚酮合酶preuA的mRNA序列全长6594-bp,如SEQID NO.2,将其均分为3段进行克隆,克隆引物见表1。
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:第一段用引物PreuA-E-1F、PreuA-E-1R;第二段引物用PreuA-E-2F、PreuA-E-2R;第三段引物用PreuA-E-3F、PreuA-E-3R。
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4(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-URA 18μL,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),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preuA基因片段1-3与1.2制备的载体片段4进行重组构建异源表达载体YEpADH2p-URA-PreuA。
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 2μL;片段4 1μL;SE Recombinase 1μL。37℃反应0.5h,冰上2min,然后转化T1PhageResistant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Axygen○R 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Eco RI/Eco RV酶切验证(7877/2530/1382/1067-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 5μL,10×FastDigest Green Buffer 1μL,异源表达质粒3μL,限制性内切酶①(Eco RI)0.5μL,限制性内切酶②(Eco RV)0.5μL。37℃酶切反应0.5h后跑胶。
2.聚酮合酶PreuA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PreuA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-PreuA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-PreuA),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,葡糖糖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,葡糖糖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 C18column(Kromasil 100-5-C18,4.6×250mm,5μm);流动相是水(A)/甲醇(B,加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3L,发酵及萃取方法分别同2.2,2.3,制得粗浸膏2.1g。将粗浸膏依次采用150mL正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇对粗浸膏进行洗涤,回收乙酸乙酯、甲醇洗涤液,浓缩后合并,即得目标单体化合物约1.5g。
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针晶,最大紫外吸收波长为320nm(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317.06784[M-H]-以及319.08127[M+H]+(图6),分子式为C16H14O7,其结构解析所需的核磁数据见图7-图8以及表2,最终解析其结构为红粉苔酸(分子结构见图1)。
3.红粉苔酸抗农作物病原真菌活性实验
3.1将红粉苔酸以及酮康唑(阳性对照)配制成1mg/mL的母液。
3.2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。
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。
3.3以酮康唑为阳性对照,取2uL目标化合物加入到198uL的目标菌液里面,采用二倍稀释法测试红粉苔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)
4.结论
实验结果表明,我们首次从光黑壳属真菌(Preussia isomera)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA,并基于酿酒酵母异源表达技术,构建了一株可高效生产红粉苔酸的酵母突变株(~0.5g/L);并且制备的红粉苔酸对苹果轮纹病菌、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,其中对苹果轮纹病菌有很强的拮抗作用(MIC,25μg/mL)。
表1:聚酮合酶基因preuA的克隆相关引物信息
表2:化合物红粉苔酸的1H与13C核磁数据(500 MHz,CD3OD,δin ppm,J in Hz)
No. | δC | δH(m,JHH,area) |
1 | 170.87,C | |
2 | 105.43,C | |
3 | 144.90,C | |
4 | 112.97,CH | 6.27(d,J=3.1,1H) |
5 | 166.73,C | |
6 | 102.02,CH | 6.20(d,J=2.4,1H) |
7 | 165.12,C | |
8 | 24.44,CH<sub>3</sub> | 2.54(s,3H) |
1’ | 174.50,C | |
2’ | 117.30,C | |
3’ | 144.75,C | |
4’ | 113.07,CH | 6.63(d,J=2.0,1H) |
5’ | 155.21,C | |
6’ | 109.41,CH | 6.60(d,J=2.3,1H) |
7’ | 164.78,C | |
8’ | 23.85,CH<sub>3</sub> | 2.56(s,3H) |
表3:红粉苔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)
聚酮分子红粉苔酸具有抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性,故可作为食品与医药行业天然抗菌剂、抗氧化剂等的潜在生物资源。但目前,市售红粉苔酸(CAS:480-56-8)均通过化学合成方式生产,售价约2000元/mg,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,我们首次从光黑壳属真菌(Preussia isomera)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA,并基于酿酒酵母异源表达技术,构建了一株可高效生产红粉苔酸的酵母突变株(~0.5g/L);并以制备的红粉苔酸为对象,研究了其对多株农作物病原真菌的拮抗作用。研究发现,红粉苔酸对苹果轮纹病菌、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,其中对苹果轮纹病菌有很强的拮抗作用(MIC,25μg/mL)。本发明极大地丰富了红粉苔酸的生产来源,对于拓展其衍生化途径,研发新型生物农药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PreuA及其在制备红粉苔酸中的应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 2
<211> 2205
<212> PRT
<213> 人工序列()
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Met Ser Asn Ser Thr Arg Asp Tyr Pro Ile Ser Ala Ala Phe Phe Cys
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Pro Gln Ser Arg Ala Pro Pro Ala Glu Tyr Leu His Ala Leu Tyr Ser
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Ser Leu Asp Glu Val Trp Pro Ile Phe Ser Glu Ala Arg Asp Asp Ile
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Leu Arg Leu Pro Asp Ala Arg Gln Asn Ile Asn Val Leu Val Asp Trp
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Ala Lys Gly Gly Ser Ser Thr Pro Ile Ala Glu Ala Arg Ser Gly Val
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Ile Ala Leu Pro Ser Val Phe Ile Val Gln Leu Gly Gln Tyr Phe Arg
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Tyr Leu Glu Ala Asn Arg Leu Ser His Gly Asp Phe Ile Gly Gln Leu
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Lys Asp Ile Gly Gly Val His Gly Tyr Cys Gly Gly Ala Ala Ala Ala
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Leu Ser Val Ala Cys Ala Ala Asp Glu Thr Gln Leu Ile Asp His Ala
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Ala Val Leu Leu Arg Leu Phe Val Gly Ile Gly Cys Cys Ile Glu Ala
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Val Asp Asp Trp Thr Thr Thr Glu Ser Thr Val Ile Ala Cys Arg Leu
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Lys Tyr Glu Gly Gln Gly Asp Glu Leu Cys Ser Arg Phe Pro Gly Thr
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Tyr Val Ser Ala Ile Thr Glu Pro Lys Ser Ile Ser Ile Thr Gly Asn
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Ala Arg Thr Leu Ser Glu Leu Phe Asp Tyr Ala Val Gly Leu Gly Leu
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Pro Thr His Lys Met Glu Ile Thr Gly Lys Ala His Asn Pro Glu Asn
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Ala Glu Leu Ala Lys Asp Phe Ile Asn Leu Tyr Arg Arg Thr Pro Ala
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Leu Gln Leu Pro Pro Thr Phe Lys Leu Gln Ala Thr Val Arg Ser Asn
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Arg Thr Ala Glu Lys Leu Thr Asn Glu Gly Ile Ile Glu Asp Met Ile
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Thr Met Ile Ile Ala Ser Gln Cys Asp Trp Asn Thr Leu Leu Thr Arg
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Val Ala Glu Asp Met Lys Val Ser Gly Arg Pro Phe His Lys Met Val
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Ser Phe Gly Met Asn Asp Cys Val Pro Val Thr Pro Phe Asn Arg Gln
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Arg Leu Lys Thr Thr Lys Phe Glu Ala His Val Leu Ile Glu Pro Leu
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Lys Pro Ser Arg Ile Ser Ala Ala Gln Tyr Pro Thr Phe Ser Asp Asp
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Ala Ile Ala Ile Thr Gly Ala Ser Leu Arg Leu Pro Gly Ala Asn Asn
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Leu Asp Glu Leu Trp Asp Leu Ile Ser Lys Gly Thr Asp Cys His Arg
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Glu Ile Pro Lys Asp Arg Phe Asp Pro His Asn Ile Tyr Arg Thr Ser
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Gln Ser Gly Phe Ser Lys Ala Gln Lys Tyr Phe Gly Asn Phe Leu Glu
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Asp Ile Lys Gly Phe Asp Arg Ala Tyr Phe Ser Met Gly Val Arg Glu
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Glu Ala Leu Glu Ala Ser Gly Tyr Leu Ala Asn His Val Arg Glu Ala
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Gly Asp Pro Val Gly Cys Phe Val Gly Ala Ser Phe Ile Glu Tyr Leu
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Glu Asn Thr Gly Ala His Pro Pro Thr Ala Tyr Thr Ala Pro Gly Thr
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Ile Arg Ala Phe Leu Cys Gly Arg Leu Ser Tyr Tyr Phe Gly Trp Thr
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Ala Pro Ala Glu Val Ile Asp Thr Ala Cys Ser Ala Ser Met Val Ala
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Ile Asn Arg Ala Val Lys Ser Ile Gln Ala Gly Glu Cys Glu Met Ala
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Leu Ala Gly Gly Val Asn Leu Ile Thr Gly Met Asn Asn Tyr Leu Asp
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Leu Ala Lys Ala Gly Phe Leu Ser Pro Thr Gly Gln Cys Lys Pro Phe
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Leu Ser Lys Ser Gly Leu Glu Pro Ala Gln Ile Thr Tyr Val Glu Ala
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His Gly Thr Gly Thr Gln Ala Gly Asp Pro Ile Glu Val Glu Ser Val
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Arg Ala Val Leu Gly Asp Pro Thr Arg Ala His Ser Leu Ser Leu Gly
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Ser Val Lys Gly Asn Ile Gly His Cys Glu Thr Gly Ala Gly Val Ala
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Leu Ala Ser His Lys Ala Leu Asn Pro Lys Ile Pro Ala Leu Glu Thr
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His His Met Glu Ile Ala Lys Gln Leu Lys Pro Trp Asp Val Pro Leu
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Arg Ala Ala Phe Val Asn Ser Tyr Gly Ala Ala Gly Ser Asn Ala Ala
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Val Ile Cys Val Glu Pro Pro Pro Val Val Thr Asp Gly Ser Ser Leu
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Ile Gly Thr Glu Pro Gln Lys Val Thr Leu Pro Val Ile Val Ser Gly
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Ala Thr Arg Lys Ser Leu Val Leu Asn Ala Arg Ala Leu Ala Ser Tyr
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Leu Ser Gln Asp Gly Ser His Leu Ser Ile His Asp Val Ala Phe Thr
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Val Asn Gln Arg Arg Lys Arg Asn Arg Phe Cys Ala Glu Val Ser Gly
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Thr Asp Leu Pro Ser Leu Val Gln Ser Leu Arg Ala Val Asp Ser Pro
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Ser Phe Glu Ser Pro Gly Lys Ser Lys Pro Val Val Leu Val Phe Ser
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Gly Gln Asn Thr Asn Ala Val Ala Leu Asp Arg Thr Ile Tyr Asp Thr
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Tyr Pro Val Phe Lys Ala Tyr Ile Asp Ala Cys Asp Ser Glu Ile Val
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Lys Leu Gly Phe Pro Ser Ile Met Glu Ala Ile Phe Gln Lys Glu Pro
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Ile Ser Thr Ala Val Ala Leu Gln Gly Ser Ile Phe Ala Met Gln Tyr
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Ala Cys Ala Arg Ser Trp Ile Asp Ala Gly Leu Lys Pro Arg Ala Ile
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Ile Gly His Ser Phe Gly Glu Leu Thr Ala Leu Ala Val Ser Gly Ala
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Leu Ser Leu Ala Asp Ser Leu Lys Leu Val Thr Cys Arg Gly His Leu
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Ile Asp Thr Lys Trp Gly Glu Glu Arg Gly Gly Met Leu Val Ile His
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Ala Asp Val Ala Thr Val Glu Arg Phe Gln Ser Arg Phe Lys Ala Gln
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Thr Val Val Ala Gly Pro Val Ala Tyr Met Asp Ala Ala Glu Gln Met
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Thr Ser Asn Ala Phe His Ser Ser Leu Ser Asp Pro Ile Leu Ala Asp
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His Leu Asn Ala Leu Ser Ala Thr Val Lys Pro Asn Pro Val Trp Leu
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Pro Pro Tyr Gln Phe Glu Arg Glu Gln His Trp Thr Glu His Ile Asp
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Ser Thr Pro Thr Gln Thr Val Gln Ser Pro Pro Lys Leu Ile Ser Arg
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Leu Ala Ser Leu Gln Tyr Gln Ile Asn Thr Gln Cys Glu Arg Phe Gln
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Lys Ile Thr Glu Gly His Ala Val Leu Tyr Glu Pro Leu Cys Pro Ala
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Ser Leu Tyr Met Glu Cys Val Val Met Ala Leu Gln Glu Leu Ala Gly
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Asp Leu Gly Ser Arg Thr Leu Asp Phe Glu Asn Leu Asp Phe His Ala
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Gly Leu Gly Leu Gln Thr Asp Arg Arg Val Leu Leu Asp Leu Glu Glu
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Ala Arg Pro His Ser Trp Thr Phe Lys Val Gln Ser Thr Lys Ala Gly
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Ser Ser Arg Ser Leu Leu His Cys Ser Gly Arg Val Ile Leu Thr Glu
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Ser Ser Val Pro Thr Thr Phe Gln Arg Leu Val Asp Gly Pro Arg Ser
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Arg Leu Asp Gln Asp Lys Asp Ala Glu Lys Leu Met Ser Ser Arg Ala
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Tyr Gly Leu Phe Ser Asn Ile Met Thr Tyr Ser Glu Phe Leu Lys Pro
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Ile Ser Ser Ile Ile Leu Arg Glu Asn Glu Ser Leu Ala Thr Ile Lys
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Leu Pro Pro Asn Gln Pro Gly Leu His Glu Ser Thr Ala Trp Lys Arg
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Cys Asp Ala Val Phe Leu Asp Gly Phe Ile Ser Ser Ser Gly Leu Leu
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Leu Asn Ser Ser Ser Val Val Gln Ser Gly His Val Leu Ile Ala Val
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Gly Val Glu Arg Ala Ile Leu Thr Ala Ala Phe Gln Ala Ser Leu Ala
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Ser Ser Trp Gln Ala Tyr Ala Thr Phe Thr Met Val Gly Glu Thr His
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Ala Leu Cys Asp Val Phe Ala Cys Thr Pro Asp Gly Glu Val Val Ala
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Met Met Thr Gly Val Arg Phe Asn Lys Met Glu Ile Ser Lys Leu Ala
1570 1575 1580
Lys Ser Leu Ser Ser Val Asn Ala Ser Ser Pro Thr Gly Gly Arg Thr
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Gln Pro Pro Ala Ala Pro Lys Thr Gln Ala Gln Pro Met Ala Ser Arg
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Pro Ser Pro Thr Pro Leu Gln Val Ser Phe Ala Thr Ala Glu Pro Ala
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Ala Pro Glu Pro Val Gln Gln Ser Thr Ala Ala Leu Ala Arg Asn Asp
1635 1640 1645
Ile Gly Pro Val Leu Lys Ser Leu Ile Ser Asn Tyr Thr Gly Leu Ile
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Glu Glu Asp Val Ser Glu Asp Ser Pro Leu Val Asp Leu Gly Leu Asp
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Ser Leu Ser Ser Val Glu Phe Ala Ser Glu Ile Gly Thr Lys Phe Gly
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Val Thr Leu Asp Ala Asp Thr Val Gly Asp Leu Thr Leu His Ser Leu
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Cys Gln Arg Leu Ser Gly Thr Ser Asn Val Val Ser Gln Lys Met Ser
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Glu Thr Pro Ala Ala Ala Pro Val Lys Glu Leu Ile Glu Thr Val Pro
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Ser Pro Ile Val Thr Phe Ser Ser Pro Val Ser Asn Ser Ile Thr Ser
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Val Leu Lys Ser Leu Leu Gly Ser Tyr Thr Gly Leu Gln Glu Glu Asp
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Met Pro Asp Asp Val Pro Leu Ile Asp Leu Gly Leu Asp Ser Leu Ser
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Ser Val Glu Phe Ala Ser Glu Leu Asn Asp Lys Met Gly Ala Asp Ile
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Asp Ser Ala Val Val Ala Asp Met Thr Leu Ser Ala Leu Glu Gln Gln
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Leu Gly Ala Ser Ala Thr Pro Pro Ser Thr Thr Gly Ser Ser Thr Pro
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Gly Asp Ile Ser Thr Ala Ala Thr Thr Pro Tyr Ala Thr Gly Ala Ser
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Thr Pro Asp Tyr Leu Val His Gly Asn Lys Pro Ser Ile Ser Asn Gly
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Val Val Ala Ala Lys Asp Ser Tyr Gln Val Lys Thr Val Glu Tyr Lys
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Arg Val Ser Gly Val Pro Ile His Ala Asp Ile Tyr Val Pro Leu Val
1890 1895 1900
Gln Arg Val Ser Pro Met Pro Leu Ala Leu Met Ile His Gly Gly Gly
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His Met Thr Leu Ser Arg Lys Ala Val Arg Pro Thr Gln Ala Lys Tyr
1925 1930 1935
Leu Leu Ser His Gly Phe Leu Pro Ile Ser Ile Asp Tyr Arg Leu Cys
1940 1945 1950
Pro Glu Val Asn Leu Ile Asp Gly Pro Ile Ala Asp Val Arg Asp Ala
1955 1960 1965
Tyr Val Trp Ala Cys Gln Asn Leu Gly Thr His Leu Ala Glu His Ser
1970 1975 1980
Ile Ser Val Asp Gly Gly Arg Val Val Val Val Gly Trp Ser Thr Gly
1985 1990 1995 2000
Gly His Leu Ala Met Ser Leu Gly Trp Ser Leu Glu Glu Ala Gly Val
2005 2010 2015
Pro Pro Pro Lys Ala Val Leu Ser Phe Tyr Ala Pro Val Asp Phe Glu
2020 2025 2030
Ser Gly Glu Leu Asp Asn Gln Lys Asn Pro Ala Leu Pro Lys Pro Arg
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Met Thr Leu Asp Gln Ile Thr Lys Ala Leu Pro Arg Thr Pro Val Thr
2050 2055 2060
Gln Tyr Gly Ala Ser Ser Thr Asp Glu Thr Asn Leu Gly Trp Leu His
2065 2070 2075 2080
Pro Gly Asp Pro Arg Ser Glu Leu Leu Leu His Val Phe His Ser Asp
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Ile Gly Leu Pro Leu Ile Leu His Gly Leu Pro Ile Ser Gly Ser Gly
2100 2105 2110
Arg Pro Ser Pro Ser Leu Val Ala Ser Ile Ser Pro Leu Ala Arg Leu
2115 2120 2125
Arg Asn Gly Ser Tyr Thr Ile Pro Thr Phe Ile Ile His Gly Thr Lys
2130 2135 2140
Asp Val Ile Ala Pro Tyr Ala Ala Ala Glu Arg Phe Val Lys Ile Met
2145 2150 2155 2160
Ser Glu Lys Gly Val Lys Ser Gly Phe Leu Ser Leu Ser Gly Thr Gly
2165 2170 2175
His Val Phe Asp Val Thr Met Lys Pro Asp Ser Lys Gly Trp Glu Asp
2180 2185 2190
Lys Val Lys Pro Gly Leu Asp Phe Leu Ile Gln Asn Ala
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<210> 1
<211> 6618
<212> DNA/RNA
<213> 人工序列()
<400> 1
atgtctaatt ctacacgtga ctatcccata tccgcggcct tcttttgtcc acagagtagg 60
gcacccccag cggaatacct ccacgccctt tattcttttc tcagccaaaa cacccttgga 120
aaggctttcc ttcgccacat tgcgtccctt gacgaagtct ggcccatctt ctccgaagcg 180
agggacgata ttctcagact gcctgatgcg cgccaaaata tcaatgtgct cgtcgattgg 240
gcaaagggtg gctcttccac tcccattgct gaagcccgat ctggagtgat cgctcttcca 300
tcagtcttca ttgtccagct tggacaatac tttcgttacc tcgaggcaaa tcggctatcc 360
cacggcgact ttatcggcca gctcaaggat attggtggtg ttcatggata ctgtggaggc 420
gctgctgcag cactctccgt tgcgtgtgca gccgatgaga cccagctcat tgaccatgct 480
gcagtgttgc tacgtttatt cgttggtatc ggctgttgca tcgaggcagt ggatgattgg 540
accacaactg agtccactgt cattgcctgc cgtctcaaat acgaaggaca gggtgatgaa 600
ctctgtagcc gatttccagg tacatacgtc tctgccatta cagagcctaa gtcaataagc 660
attactggca atgcccgcac actgtcagag ctttttgatt atgcggtggg ccttggactt 720
cccacccata agatggaaat caccggtaaa gcacacaacc cagaaaacgc tgaactggcc 780
aaagatttta tcaacttata tcgtcggact ccggctctgc aactgccccc taccttcaag 840
ttgcaagcaa cagtgcgctc aaatcgtact gcggagaagt tgaccaacga aggcattatt 900
gaggacatga tcacgatgat tatagcgtcc caatgtgact ggaacacgct tctgacaaga 960
gtcgccgagg acatgaaggt ctctggtcga ccatttcaca agatggtgtc ctttgggatg 1020
aacgattgcg ttcctgtaac acctttcaat cgacagcggc ttaagaccac caaatttgag 1080
gctcatgtcc tcatcgagcc cctgaagcct tcgcgtattt ccgctgcgca gtatcctacc 1140
ttctcagatg atgcaatcgc cataacaggc gcttctttgc gcctaccagg tgcaaataat 1200
cttgacgaat tatgggacct gatctccaaa ggtaccgact gtcacaggga aataccaaaa 1260
gacagattcg atccgcacaa catttatcgg acctctcaga gtggcttcag caaagcccag 1320
aagtattttg gcaactttct tgaggacatc aaagggttcg atagggcgta tttcagcatg 1380
ggtgtacggg aagctgccaa catcgaccca caacagcgat tactcctgga gcttgcagtt 1440
gaagcccttg aggcaagtgg ctatctcgcc aaccatgtac gagaagctgg tgacccggtc 1500
ggctgctttg ttggagccag ctttatagaa tacctggaaa atactggtgc ccaccctcca 1560
acagcttaca ccgctcccgg aaccatcaga gcctttttat gtggcagact cagctattac 1620
tttggatgga cagccccagc ggaagttatc gatactgcct gctcggcttc catggtcgcg 1680
atcaaccgcg cagtcaaatc tatccaagca ggagaatgtg agatggcgct tgctggaggc 1740
gtaaacctga tcactggaat gaacaactat ctcgatttgg ccaaagccgg atttctgagc 1800
ccaacaggcc aatgcaagcc attcgaccaa tctggagatg gctattgtcg ctctgatgga 1860
gcaggatttg ttgttctgaa gaagttgtcg caggctctgg taaatggcga tccgatcatg 1920
ggtgttattc ccagtatcga aaccaatcaa ggtggtctat ccgggtcact cactgttcca 1980
tcatccactg cactacaagc actttacaaa cgcgtccttt cgaaatctgg tctggaacct 2040
gcacagatta cctatgttga agcccatggt acaggaaccc aagcaggtga cccgattgag 2100
gtggagagcg ttcgtgcagt tctcggagac cccacgcgag cccattctct ttccctaggc 2160
tctgtgaaag gaaacattgg ccattgcgaa actggcgctg gcgtcgctgg tctgctgaaa 2220
gtacttgcaa tgatcaaaca tggaggtatc ccgcctctgg caagccacaa agcgctcaat 2280
cctaaaatac cagccttgga gacacatcac atggaaattg caaagcagct taagccttgg 2340
gatgtcccac tgagggcagc atttgtcaac agttatggcg ctgctggctc gaatgctgcc 2400
gtgatctgtg tcgagccacc accagtcgtc accgacggat catctttgat tggcactgag 2460
cctcaaaagg taacgctccc agtcatcgtc agtggcgcta caagaaagag cttggtcctg 2520
aatgcacgag cattggcaag ctacctctcg caagacggat cacacctcag catacatgat 2580
gttgcgttta ccgtcaacca acggagaaaa cggaatagat tttgcgccga ggtctctggc 2640
accgatttgc catctttggt tcagtcactc cgcgctgtag actctcccag tttcgagagc 2700
cctggaaagt ccaagcctgt ggtgctcgtc ttcagcggac aaaacaccaa tgcagtagcc 2760
ttggaccgca cgatatacga tacctaccca gtatttaaag cctatattga cgcctgtgac 2820
tccgaaattg tgaaacttgg cttcccaagt atcatggagg ccattttcca gaaggagccc 2880
ataagcactg ctgttgcttt gcaaggcagt atcttcgcaa tgcagtatgc gtgtgcccgt 2940
tcttggatcg acgcaggcct caaacctcga gcaatcatcg gccacagttt cggcgaactt 3000
actgctttgg ctgtctctgg agctctgtca cttgcagaca gcttgaagct ggtcacatgc 3060
cgcggtcacc tcatcgacac taagtggggt gaggaaaggg gcggtatgct tgtcattcat 3120
gcggatgtgg ccacggtcga acgcttccaa tcccggttca aagcgcagca tgacggagct 3180
gaactggaga ttgcttgcta taactctcca actaccacag tggttgctgg gccagtggca 3240
tacatggatg cagctgagca gatgctggcc acagatccag atttccaggg ccttcgtaag 3300
ctgcgcattg taacgagtaa cgccttccat tcttcgttgt cggatccgat cctagccgat 3360
ttggactcca tggcagatac cttgacctgg aatgagccca gtatcccact ggaagcttgc 3420
acgagtgaag gcctggcgtc gatcaaagag tggagtgcct ccagacatac cagaggctca 3480
gtgtacttca ccaaagcggt ggagcgtatc gaaggacgac tgggtgcatg catctgggtt 3540
gaagccggcc ttgactccgc catcatagca atggctcgaa aagcttcttc aaagccagac 3600
tcgcaggtct ttcaatctgt cagcacgaag gctggagcta cttctttcat tgatggaatc 3660
gtaaacaatc tttggcgtca aggcgtgcct ctttctcact tgaacgcgct atcggcgact 3720
gtcaaaccca accctgtctg gcttccacca taccagttcg aaagagaaca gcattggacc 3780
gagcacatcg atagggcgac cgaagcaagt caagcaagca ccacaagcga cactattcag 3840
tcgactccaa cgcaaaccgt ccaaagtcca ccaaagctga tttccagact cgcatctctg 3900
cagtatcaga tcaacacgca gtgcgagcgc ttccaaaaga tcaccgaagg ccatgcggtg 3960
ttgtatgaac ccttatgccc tgcatccttg tacatggaat gtgttgtcat ggctctccaa 4020
gaactagcag gcgaccttgg ttcccgcact cttgacttcg aaaatctgga cttccatgcg 4080
gggttgggcc tacagactga ccgccgtgtg cttctcgact tggaagaagc gcgccctcac 4140
tcatggactt tcaaggtgca atccacgaaa gctgggtcat ctcggtcatt gttacattgc 4200
tcgggccggg tgatcctaac tgagagttcg gtacctacca cgttccagcg tctggtcgat 4260
ggccccagat cacgtctcga ccaggacaag gatgctgaga agcttatgtc gtctcgcgcc 4320
tacggtctgt tttccaacat catgacctat tcggaattcc tcaagccgat ttcgtcgatc 4380
atcctgcgcg aaaacgaatc cttggctacc atcaaacttc caccgaacca gcctggtctg 4440
catgaaagca cggcttggaa aagatgcgac gcagtgttcc tggatggctt catctcctct 4500
tcgggacttc tgctcaatag cagcagcgtg gttcagtcag gccatgtttt gatcgctgtt 4560
ggagtcgagc gggccatcct cacagctgct ttccaagcat cccttgcttc ctcatggcag 4620
gcgtacgcaa catttaccat ggttggcgaa actcacgctc tctgcgatgt tttcgcttgc 4680
actcctgacg gtgaagtggt agccatgatg acaggcgtga ggttcaacaa gatggagatc 4740
tcaaagttag cgaaatcgct ctcgtcggtc aacgcctcgt caccgacagg tggaagaact 4800
cagccaccag ccgcgccaaa aacccaagct cagccgatgg cttctagacc ctcacctacc 4860
ccactgcagg tttcctttgc aacggcagaa cctgccgcac cagagcctgt tcaacagtca 4920
acagctgccc tagcgcgtaa tgacataggt ccagtcctta agtctctcat ctccaactac 4980
accggcctaa tcgaagagga tgtctcggaa gatagtcctc tcgtcgactt aggtcttgac 5040
tcgctctctt ccgttgagtt cgcgtctgaa atcggaacca agttcggagt cactctggat 5100
gcggatacag tgggagactt gacgttacac tcgctttgcc agaggctcag tggcacctcg 5160
aacgttgtat cccagaagat gtccgagaca cctgcagcgg ctccggtgaa ggagctgatt 5220
gaaactgtac catcacccat cgtgactttc agcagtcctg tgtcaaacag catcacatcg 5280
gtcctaaagt ctcttctcgg gagctacacc ggcttacaag aagaagacat gcccgacgat 5340
gtacctctta ttgatcttgg actggattcc ttgtcatccg tcgagtttgc gtcggaactg 5400
aacgacaaaa tgggagcaga tatcgactcc gctgttgttg cagacatgac cttatctgcg 5460
ctcgagcaac agctgggagc atctgcgaca ccacctagca ccaccggctc atccactcct 5520
ggggatattt caactgctgc cacgacccct tatgctacgg gagcctctac gccagactac 5580
ttggtgcatg gaaataaacc gtctatttca aatggagttg tcgctgcaaa agactcctat 5640
caagtcaaga ccgtcgaata caaacgggta agtggtgttc ccatccacgc agacatctac 5700
gtccctcttg ttcaacgcgt ctctcctatg cccttagcgc tcatgatcca cggcggtggc 5760
cacatgacct tgtctcggaa ggccgtccgt cccacacaag ccaaatatct tctgtcgcac 5820
ggcttcctcc ccattagtat tgactaccgt ctgtgtccgg aggtcaatct catcgatgga 5880
cccatcgccg acgtacgtga tgcttatgtg tgggcttgtc aaaatctggg gacgcatctt 5940
gcggaacaca gtatctctgt cgatggtggc cgcgtggtgg tggtggggtg gtcgactgga 6000
ggtcatttgg caatgtcact gggatggtcg cttgaagagg ctggcgtgcc gccaccaaag 6060
gctgtgttga gcttctatgc cccagtggat tttgagtctg gtgagctcga taatcaaaag 6120
aaccccgctc tcccgaaacc gcgcatgact ttggaccaaa ttacaaaggc acttcctagg 6180
accccagtga cgcagtatgg tgcctcttcc acggacgaaa caaatctcgg atggctgcac 6240
ccaggcgatc cgcgatccga actccttctc cacgtgttcc actccgatat tggcttaccg 6300
ctcatactac acggtcttcc catttcgggt tctggcaggc catcgccttc ccttgttgct 6360
tccataagtc cgctggcgcg cttgcgcaac ggctcataca ctatcccgac cttcatcatt 6420
catggcacga aggacgtcat cgctccatat gcagcagcgg agcgatttgt caagattatg 6480
tccgagaaag gggtaaagag tggatttcta tcgttgtcag ggacgggtca tgttttcgat 6540
gttacgatga agcctgatag taaggggtgg gaggacaagg ttaagccggg gcttgacttt 6600
ttgattcaga acgcttga 6618
<210> 3
<211> 60
<212> DNA
<213> 人工序列()
<400> 3
gactacaaag acgatgacga caagcttcat atgtctaatt ctacacgtga ctatcccata 60
<210> 4
<211> 52
<212> DNA
<213> 人工序列()
<400> 4
catgtttgat cattgcaagt actttcagca gaccagcgac gccagcgcca gt 52
<210> 5
<211> 43
<212> DNA
<213> 人工序列()
<400> 5
gctgaaagta cttgcaatga tcaaacatgg aggtatcccg cct 43
<210> 6
<211> 25
<212> DNA
<213> 人工序列()
<400> 6
tcggcttgag gaattccgaa taggt 25
<210> 7
<211> 56
<212> DNA
<213> 人工序列()
<400> 7
tcatgaccta ttcggaattc ctcaagccga tttcgtcgat catcctgcgc gaaaac 56
<210> 8
<211> 59
<212> DNA
<213> 人工序列()
<400> 8
agtgatggtg atggtgatgt ccgtttaaac tcaagcgttc tgaatcaaaa agtcaagcc 59
Claims (9)
1.聚酮合酶PreuA,其特征在于,所述聚酮合酶PreuA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PreuA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PreuA在制备红粉苔酸中的应用。
7.一株可高效生产红粉苔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.红粉苔酸的制备方法,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,得到红粉苔酸。
9.根据权利要求8所述的红粉苔酸的制备方法,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:依次采用正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇对粗浸膏进行洗涤,回收乙酸乙酯、甲醇洗涤液,浓缩后合并,即得;其中,所使用的正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇的体积为SC-Ura液体培养基体积的十分之一。
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