CN113604448B - 聚酮合酶Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用 - Google Patents

聚酮合酶Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用 Download PDF

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CN113604448B
CN113604448B CN202110910678.3A CN202110910678A CN113604448B CN 113604448 B CN113604448 B CN 113604448B CN 202110910678 A CN202110910678 A CN 202110910678A CN 113604448 B CN113604448 B CN 113604448B
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刘庆培
杨小龙
张丹
徐瑶
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Abstract

本发明涉及一种聚酮合酶Preu3及其在制备2,4‑二羟基‑3,6‑二甲基苯甲酸中的应用,属于微生物化学技术领域。本发明首次克隆到与合成橡苔原料——2,4‑二羟基‑3,6‑二甲基苯甲酸合成相关的聚酮合酶Preu3,并基于异源表达技术,构建了可高效生产2,4‑二羟基‑3,6‑二甲基苯甲酸的酿酒酵母突变株;并以制备的2,4‑二羟基‑3,6‑二甲基苯甲酸为对象,研究了其对八种临床耐药细菌的抑制活性,发现其对耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌以及多耐药性屎肠球菌均有很强的拮抗作用。本发明工艺简单,产量高,对于生产合成橡苔以及研发新型抗菌剂具有重要的科学价值和应用前景。

Description

聚酮合酶Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中 的应用
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Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用。
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——聚酮合酶(polyketidesynthase,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。
2,4-二羟基-3,6-二甲基苯甲酸(2,4-dihydroxy-3,6-dimethylbenzoic acid,结构见图1),多从天然地衣中分离,在真菌中首次从土曲霉(Aspergillus terreus)中分离。经研究报道,2,4-二羟基-3,6-二甲基苯甲酸及其酯类衍生物具有抗菌、抗肿瘤、抑制乙酰胆碱酯酶、拮抗雄激素受体等生物活性,故其可作为食品与医药行业天然抗菌剂、治疗前列腺癌药物等的潜在生物资源。值得一提的是,2,4-二羟基-3,6-二甲基苯甲酸也是地衣香料——合成橡苔(2,4-二羟基-3,6-二甲基苯甲酸甲酯)的重要化学合成原料。但目前,市售2,4-二羟基-3,6-二甲基苯甲酸(CAS:4707-46-4)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高;且近年来研发的2,4-二羟基-3,6-二甲基苯甲酸的合成工艺也相对复杂,如CN109666707A、CN111116370A。因此如何克服2,4-二羟基-3,6-二甲基苯甲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,提供一种聚酮合酶Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用。
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Preu3,所述聚酮合酶Preu3的氨基酸序列如SEQ IDNO.1所示。
本发明第二方面提供编码上述聚酮合酶Preu3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一种用上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上述聚酮合酶Preu3在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用。
本发明第六方面提供一株可高效生产2,4-二羟基-3,6-二甲基苯甲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2,4-二羟基-3,6-二甲基苯甲酸的制备方法,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2,4-二羟基-3,6-二甲基苯甲酸。
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:将粗浸膏用少量甲醇溶解后,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,共得到七个组分段Fr.A-Fr.G;目标代谢物2,4-二羟基-3,6-二甲基苯甲酸集中在Fr.G组分中,将Fr.G组分通过高效液相制备得到2,4-二羟基-3,6-二甲基苯甲酸;
其中,梯度洗脱流动相采用的甲醇:水的体积比依次为5:95、20:80、30:70、35:65、40:60、45:55、50:50,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。
本发明第八方面提供上述2,4-二羟基-3,6-二甲基苯甲酸的制备方法制得的2,4-二羟基-3,6-二甲基苯甲酸在制备抗临床耐药细菌药物中的应用,其特征在于,所述的临床耐药细菌为耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、耐碳青霉烯肺炎克雷伯菌、耐碳青霉烯大肠埃希菌。
本发明中将粗浸膏用少量甲醇溶解,此处对于甲醇的用量没有具体限制,甲醇用量以粗浸膏可溶解的量即可。
在本发明中,我们首次从光黑壳属真菌(Preussia isomera)中克隆到与2,4-二羟基-3,6-二甲基苯甲酸(合成橡苔原料)合成相关的聚酮合酶Preu3,并基于酿酒酵母异源表达技术,构建了可高效生产2,4-二羟基-3,6-二甲基苯甲酸的酵母突变株(2,4-二羟基-3,6-二甲基苯甲酸产量约为0.1g/L);并以制备的2,4-二羟基-3,6-二甲基苯甲酸为对象,研究了其对八种临床耐药细菌的抑制活性。研究发现,2,4-二羟基-3,6-二甲基苯甲酸对耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌以及多耐药性屎肠球菌三种临床耐药细菌均有很强的拮抗作用(MIC,25μg/mL)。通过本发明极大地丰富了2,4-二羟基-3,6-二甲基苯甲酸的生产来源,相较于纯化学方法生产,工艺简单,产量高,绿色环保,对于拓展其衍生化途径,生产合成橡苔,以及研发新型抗菌剂均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2,4-二羟基-3,6-二甲基苯甲酸(CAS:4707-46-4)均通过化学合成方式生产,售价约1000元/100mg,产量低,售价高。本发明基于异源表达技术,构建了一株可高效生产2,4-二羟基-3,6-二甲基苯甲酸的酵母突变株(~0.1g/L),提供了新的微生物发酵途径来生产合成橡苔原料,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2,4-二羟基-3,6-二甲基苯甲酸对八种临床耐药细菌(耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、耐碳青霉烯肺炎克雷伯菌、耐碳青霉烯大肠埃希菌)的抑制活性。研究发现,2,4-二羟基-3,6-二甲基苯甲酸对耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌以及多耐药性屎肠球菌均有很强的拮抗作用(MIC,25μg/mL),有望研发新型抗生素。
附图说明
图1为2,4-二羟基-3,6-二甲基苯甲酸(2,4-dihydroxy-3,6-dimethylbenzoicacid)的化学结构式;
图2为聚酮合酶Preu3异源表达载体YEpADH2p-URA-Preu3的质粒图谱;
图3为聚酮合酶Preu3异源表达载体YEpADH2p-URA-Preu3的酶切验证图;其中,M,Trans 2K plus marker;1和2均为载体YEpADH2p-URA-Preu3;
图4为聚酮合酶Preu3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;
图5为聚酮合酶Preu3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2,4-二羟基-3,6-二甲基苯甲酸的1H核磁光谱图(氘代甲醇,500MHz);
图8为化合物2,4-二羟基-3,6-二甲基苯甲酸的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。
本发明除非另有说明,否则百分号为质量百分数。
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)中克隆到与聚酮分子2,4-二羟基-3,6-二甲基苯甲酸(化合物分子结构见图1)合成相关的聚酮合酶Preu3,并基于酿酒酵母异源表达技术,构建了一株可高效生产2,4-二羟基-3,6-二甲基苯甲酸的酵母突变株。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Preu3的异源表达载体YEpADH2p-URA-Preu3(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2,4-二羟基-3,6-二甲基苯甲酸(~0.1g/L);最后,以制备的2,4-二羟基-3,6-二甲基苯甲酸为对象,研究了其对八种临床耐药细菌(耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、耐碳青霉烯肺炎克雷伯菌、耐碳青霉烯大肠埃希菌)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Preu3异源表达载体YEpADH2p-URA-Preu3的构建
1.1聚酮合酶基因preu3的克隆:聚酮合酶preu3的mRNA序列全长7572-bp,如SEQID NO.2,将其均分为3段进行克隆,克隆引物见表1。
表1:聚酮合酶基因preu3的克隆相关引物信息
Figure BDA0003203492190000031
Figure BDA0003203492190000041
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Preu3-E-1F、Preu3-E-1R;第二段引物用Preu3-E-2F、Preu3-E-2R;第三段引物用Preu3-E-3F、Preu3-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)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-Preu3。
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T1 PhageResistant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
Figure BDA0003203492190000042
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酶切验证(7609/4773/992/460-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 7μL,10×FastDigest Green Buffer 1μL,异源表达质粒1.5μL,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),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 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1.1g。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)分别为5:95、20:80、30:70、35:65、40:60、45:55、50:50,各梯度洗脱所用的流动相均为100mL),共得到七个组分段(Fr.A-G),其中目标代谢物集中在Fr.G组分中,将其通过高效液相制备(色谱条件同2.3,tR=25.6min)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:目标代谢产物为浅黄色晶体,最大紫外吸收波长为310nm(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181.05035[M-H]-以及183.06523[M+H]+(图6),分子式为C9H10O4,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2,4-二羟基-3,6-二甲基苯甲酸(分子结构见图1)。
表2:化合物2,4-二羟基-3,6-二甲基苯甲酸的1H与13C核磁数据(500MHz,CD3OD,δin ppm,J in Hz)
No. δC δH(m,J<sub>HH</sub>,area)
1 111.40,C
2 163.28,C
3 108.95,C
4 158.64,C
5 110.09,CH 6.10(s,1H)
6 140.95,C
7 177.58,C
8 8.42,CH<sub>3</sub> 1.99(s,3H)
9 23.83,CH<sub>3</sub> 2.50(s,3H)
3.2,4-二羟基-3,6-二甲基苯甲酸抗临床耐药细菌活性实验
3.1将2,4-二羟基-3,6-二甲基苯甲酸以及环丙沙星(阳性对照)用DMSO配制成1mg/mL的母液。
3.2将目标临床耐药细菌(耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌、多耐药性屎肠球菌、多耐药性粪肠球菌、耐碳青霉烯铜绿假单胞菌、耐碳青霉烯鲍曼不动杆菌、耐碳青霉烯肺炎克雷伯菌、耐碳青霉烯大肠埃希菌)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。
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。
3.3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2,4-二羟基-3,6-二甲基苯甲酸对以上八种临床耐药细菌的拮抗活性。(活性结果见表3)
表3:2,4-二羟基-3,6-二甲基苯甲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)
Figure BDA0003203492190000061
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2,4-二羟基-3,6-二甲基苯甲酸合成相关的聚酮合酶Preu3,并基于酿酒酵母异源表达技术,构建了一株可高效生产2,4-二羟基-3,6-二甲基苯甲酸的酵母突变株(~0.1g/L);并且制备的2,4-二羟基-3,6-二甲基苯甲酸对耐甲氧西林金黄色葡萄球菌、多耐药性表皮葡萄球菌以及多耐药性屎肠球菌均有很强的拮抗作用(MIC,25μg/mL)。
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及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用
<160> 8
<170> SIPOSequenceListing 1.0
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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 Val Asn Lys Val
1780 1785 1790
Ala Gln Arg Thr Phe Ser Asp Ile Arg Pro Lys Tyr Asp Val Phe Ala
1795 1800 1805
Ala Glu Glu Gly Phe Ala Gly Phe Trp Arg Asp Val Tyr Pro Arg Gln
1810 1815 1820
Lys Arg Leu Thr Leu Ala Tyr Val Val Glu Ala Phe Ala Val Met Gly
1825 1830 1835 1840
Cys Asp Leu Ser Asp Leu Ala Ala Gly Gln Ile Leu Pro Lys Ile Glu
1845 1850 1855
Tyr Leu Pro Gln His Val Ser Leu Ile Lys Gln Leu Tyr Val Ile Leu
1860 1865 1870
Ala Asp Ser Thr Leu Ile Thr Ile Glu Asn Gly Thr Tyr Arg Arg Thr
1875 1880 1885
Arg Val Ser Val Asp Thr Thr Pro Ala Ser Asp Leu Leu Ala Asp Ile
1890 1895 1900
Leu Arg Ala Phe Pro Gln His Ala Glu Glu His Arg Leu Leu Asp Val
1905 1910 1915 1920
Thr Gly Ser Arg Leu Gly Asp Cys Leu Ile Gly Arg Ala Asp Pro Leu
1925 1930 1935
Arg Leu Leu Phe Met Asp Arg Ala Asn Lys Glu Leu Leu Asp Ser Val
1940 1945 1950
Tyr Ala Asn Gly Pro Met Tyr Lys Ala Met Ser Arg Leu Leu Gly Ser
1955 1960 1965
Tyr Ile Leu Asp Thr Met Gln Gln Trp Gln Gly Gln Lys Pro Leu Arg
1970 1975 1980
Ile Leu Glu Ile Gly Gly Gly Thr Gly Gly Thr Thr Lys His Ile Val
1985 1990 1995 2000
Lys Leu Leu His Gln Gln Gly Ile Asp Phe Thr Tyr Cys Phe Ser Asp
2005 2010 2015
Leu Ser Arg Ala Leu Val Thr Lys Ala Lys Lys Thr Phe Ser Ile Tyr
2020 2025 2030
Pro Gln Met Glu Tyr Met Val Leu Asp Ile Glu Ala Pro Pro Ser Ser
2035 2040 2045
Glu Tyr Leu Gly Gln Phe Asp Leu Ile Leu Ser Thr Asn Cys Ile His
2050 2055 2060
Ala Thr Lys Asn Ile Gln Gln Thr Thr Lys His Met Arg Gln Leu Leu
2065 2070 2075 2080
Ser Ser Glu Gly Phe Ile Cys Leu Val Glu Phe Thr Arg Asn Ile Phe
2085 2090 2095
Trp Phe Asp Leu Val Phe Gly Leu Leu Asp Gly Trp Trp Leu Phe Glu
2100 2105 2110
Asp Gly Arg Pro His Val Leu Ala Asp Glu Asn Leu Trp Asp Gln Ser
2115 2120 2125
Leu Arg Ala Ala Gly Tyr Gly Asp Val Gln Trp Thr Glu Gly Gln Ser
2130 2135 2140
Glu Glu Ser Lys Thr Leu Arg Leu Ile Ala Ala Phe Asn Val Ser Asn
2145 2150 2155 2160
Glu Asp Ala Lys Ala Ala Asn Ala Leu Ala Ser Ala Leu Ala Val Pro
2165 2170 2175
Gly Arg Lys Gly Arg Thr Ser Ala Thr Thr Ile Arg Trp Lys Gln Glu
2180 2185 2190
Gly Asp Leu Asp Leu Met Ala Asp Val Tyr Leu Pro Ser Asp Leu Asp
2195 2200 2205
Ala Ser Thr Val Ser Arg Pro Val Ala Leu Ile Leu His Gly Gly Gly
2210 2215 2220
His Val Leu His Thr Arg Lys His Ile Asn Pro Arg His Ile Lys Met
2225 2230 2235 2240
Leu Gln Asp Leu Gly Phe Leu Pro Val Ser Val Asp Tyr Arg Leu Cys
2245 2250 2255
Pro Glu Val Asn Ile Arg Asp Gly Pro Met Thr Asp Ala Cys Glu Ala
2260 2265 2270
Val Asp Trp Ala Arg Asn Ile Leu Pro Cys Leu Pro Val Cys Ser Glu
2275 2280 2285
Leu Arg Val Asp Lys Glu His Val Val Val Ile Gly Tyr Ser Thr Gly
2290 2295 2300
Gly His Leu Ala Leu Thr Thr Ala Phe Thr Thr Arg Val Arg Gly Phe
2305 2310 2315 2320
Lys Pro Pro Ser Ala Ile Leu Gly Phe Tyr Cys Pro Thr Asn Tyr Ser
2325 2330 2335
Ala Asp Trp Trp Arg Ser Pro Ile Tyr Pro Glu Leu Ala Gln Gln Ser
2340 2345 2350
Ser Ser Glu Thr Phe Asp Leu Leu Glu Gly Val Asn Glu His Ala Ile
2355 2360 2365
Ala Gly Tyr Thr Pro Thr Val Asn Asn Asn Val Ala Ala Leu Leu Met
2370 2375 2380
Ser Leu Asp Asp Pro Arg Trp Arg Phe Val Leu His Ala Asn Trp Arg
2385 2390 2395 2400
Ala Gln Thr Leu Pro Met Leu Ile Asn Gly Leu Pro Ser Lys Ser Arg
2405 2410 2415
Leu Ala Arg Ser Gly Gln Thr Val Asp Ser Val Ile Asn Arg Glu Ile
2420 2425 2430
Pro Asp Ala Glu Asp Val Ala Ser Ile Ser Pro Tyr Asp Gln Ile Val
2435 2440 2445
Arg Gly Ser Tyr Ser Thr Pro Thr Phe Leu Leu His Gly Thr Lys Asp
2450 2455 2460
Asp Leu Ile Pro Trp Gln Gln Ser Ile Ala Thr Val Asp Ala Leu Ala
2465 2470 2475 2480
Arg Arg Gly Val Asn Ala Arg Val Glu Ile Ile Glu Gly Ala Glu His
2485 2490 2495
Cys Phe Asp Val Trp Ser Asp Lys Tyr Asp Gly Met Ile Gly Arg Ala
2500 2505 2510
Leu Glu Trp Leu Val Glu Gln Cys Arg Asn Ala
2515 2520
<210> 2
<211> 7572
<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 ccctgtcaac aaagtcgctc aacggacatt ctcagacatt 5400
cgccctaagt acgacgtatt tgcagcagaa gaagggttcg ccggcttctg gcgcgacgtt 5460
taccctagac agaagcgtct cacacttgcc tatgtagtcg aggcttttgc agtgatgggg 5520
tgcgacctta gcgatcttgc cgctggacag atactcccaa agatagagta tttgccacaa 5580
catgtgagcc tcatcaagca gctctatgtt atcctggccg attcgaccct gatcacgatc 5640
gagaacggaa cctatcgcag aacacgtgta tccgtcgata ctacacctgc ttcggattta 5700
ctggcagata tcctacgtgc gttcccacag catgcagagg aacaccgact gctggatgtc 5760
actggttcgc gcctagggga ctgtctcatt ggccgcgcag atccgttacg gcttttgttc 5820
atggacagag ctaacaagga acttctcgac agcgtctatg caaatggtcc tatgtacaaa 5880
gctatgagcc gactcctggg ctcatacatt ctcgacacaa tgcaacaatg gcaaggacaa 5940
aaaccgctac gtattctcga gatcggcgga gggaccggcg gtaccacgaa gcatatcgtc 6000
aaacttctcc atcaacaggg gattgacttc acatactgtt tcagcgactt gtcacgtgcg 6060
cttgttacga aggcaaagaa aacattttcg atctatccgc aaatggaata catggtcctg 6120
gatattgaag caccaccgtc cagcgaatac cttggccaat tcgacctcat cttgtctacg 6180
aactgcatcc atgccacgaa aaatattcag cagactacaa agcatatgcg ccaactcctg 6240
agttccgaag ggttcatctg cttggtggaa tttacaagga atatcttctg gttcgatctg 6300
gtgttcggct tgctggacgg atggtggctc tttgaagacg gacgaccaca cgttcttgct 6360
gacgaaaatc tctgggacca gagcttacga gcagctggtt atggagacgt gcagtggact 6420
gaagggcagt ctgaggaatc caagacgcta cgactcattg ctgcgttcaa cgtaagcaac 6480
gaggatgcga aggcagcgaa cgctctggcg agcgccttgg ccgtacccgg gcgtaaaggc 6540
aggaccagtg cgacgaccat acgctggaag caagagggcg atctggatct catggcggat 6600
gtatacctac cgtctgatct ggatgcatca actgtaagca gacccgtcgc tctgatcctt 6660
cacggtggcg gccatgtctt gcacactaga aaacacatca acccgcgaca catcaaaatg 6720
ctacaagatc ttggcttctt accggtttca gtcgattatc gtctttgtcc agaggtcaac 6780
attcgcgatg gacctatgac ggacgcatgt gaggcggtgg actgggcaag gaatattcta 6840
ccatgtctgc ctgtgtgctc cgagttgcgt gtcgacaaag agcacgtagt ggtgattggc 6900
tactctacag gcggacacct tgctctgaca actgcattca caacacgtgt caggggattc 6960
aagcccccct ctgccattct gggattctac tgcccgacca actatagcgc cgactggtgg 7020
cggtctccca tctatccgga gcttgctcag cagtctagct cggagacttt cgacttgctg 7080
gaaggtgtca atgagcatgc gattgctgga tacacaccaa cagtgaataa taatgtcgct 7140
gcgcttctga tgtctcttga cgacccacgc tggcgcttcg tcctacatgc caactggcga 7200
gctcagactc tcccgatgct tatcaacggg ctgccctcta agtctcggct tgcgcgcagt 7260
ggacaaacgg tggacagtgt catcaacagg gagattccag acgctgaaga tgttgcgtct 7320
atcagcccgt atgatcaaat cgtcagagga agctacagta cccctacatt ccttctccat 7380
ggaacgaagg acgatctcat tccttggcag cagagcatag cgacagttga tgctctggca 7440
cgacgtgggg tgaatgcgag agtggagatt attgagggtg cggagcactg ttttgatgtt 7500
tggtctgata agtatgatgg gatgattggg agggcactgg agtggttggt ggagcagtgt 7560
cggaatgctt ag 7572
<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> 58
<212> DNA
<213> 人工序列()
<400> 8
agtgatggtg atggtgatgt ccgtttaaac ctaagcattc cgacactgct ccaccaac 58

Claims (9)

1.聚酮合酶Preu3,其特征在于,所述聚酮合酶Preu3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Preu3在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用。
7.一株可高效生产2,4-二羟基-3,6-二甲基苯甲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。
8.2,4-二羟基-3,6-二甲基苯甲酸的制备方法,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2,4-二羟基-3,6-二甲基苯甲酸。
9.根据权利要求8所述的2,4-二羟基-3,6-二甲基苯甲酸的制备方法,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:将粗浸膏用少量甲醇溶解后,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,共得到七个组分段Fr.A-Fr.G;目标代谢物2,4-二羟基-3,6-二甲基苯甲酸集中在Fr.G组分中,将Fr.G组分通过高效液相制备得到2,4-二羟基-3,6-二甲基苯甲酸;
其中,梯度洗脱流动相采用的甲醇:水的体积比依次为5:95、20:80、30:70、35:65、40:60、45:55、50:50,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。
CN202110910678.3A 2021-08-09 2021-08-09 聚酮合酶Preu3及其在制备2,4-二羟基-3,6-二甲基苯甲酸中的应用 Active CN113604448B (zh)

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