WO2020020188A1 - 一种新型异戊烯转移酶及其应用 - Google Patents

一种新型异戊烯转移酶及其应用 Download PDF

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WO2020020188A1
WO2020020188A1 PCT/CN2019/097372 CN2019097372W WO2020020188A1 WO 2020020188 A1 WO2020020188 A1 WO 2020020188A1 CN 2019097372 W CN2019097372 W CN 2019097372W WO 2020020188 A1 WO2020020188 A1 WO 2020020188A1
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isopentenyl
transferase
isopentenyl transferase
gene
donor
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李文利
姚婷婷
刘晶
李花月
李通
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中国海洋大学
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Priority to US17/042,942 priority Critical patent/US11279918B2/en
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Priority to US17/589,494 priority patent/US11680251B2/en

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  • the invention belongs to the fields of genetic engineering and biopharmaceutical technology, and particularly relates to a novel phytoene-synthase-like (PSL) family of isopentenyl transferases and applications thereof.
  • PSL phytoene-synthase-like
  • Isopentenyl is widely present in many active natural products, such as alkaloids, flavones, and other compounds.
  • the introduction of isopentenyl in natural products not only greatly enriched the structural diversity of the compound, but also increased its affinity with drug targets and bioavailability due to its increased lipophilicity, and improved pharmacokinetic characteristics, thereby improving Medicine.
  • a large number of structure-activity relationship studies have shown that the biological activity of isopentenyl aromatic compounds originates from the isoprenyl groups substituted on the backbone of aromatic compounds. Due to the complex and diverse structure of natural products, it is difficult to carry out isopentenylation by chemical methods.
  • the use of a substrate broad-spectrum isopentenyl transferase for isopentenylation provides a new strategy for this.
  • Aromatic isopentenyl transferase can integrate isoprene units into compounds containing aromatic rings, thereby forming active molecules with multiple biological functions in the primary or secondary metabolism of an organism.
  • the phytoene-synthase-like (PSL) family of isopentenyl transferases is a new class of isopentenyl transferases based on compounds containing aromatic rings as a substrate. Bioinformatics analysis shows that they are octahydrolycopene synthase It is farther away from the typical aromatic isopentenyl transferase.
  • PSL family transferase is NzsG derived from the neocarazostatin biosynthesis process in Streptomyces sp. MA37, but it has a very strong substrate specificity and can only transfer dimethylallyl pyrophosphate (DMAPP) to precarazostatin.
  • DMAPP dimethylallyl pyrophosphate
  • the object of the present invention is to provide a new type of phytoene-synthase-like (PSL) family of isopentenyl transferases, which has a broad spectrum of substrates, and can use a variety of cyclic dipeptides and isopentenyl as substrates to produce multiple Drimentines precursor compounds.
  • PSL phytoene-synthase-like
  • the present invention first provides a new type of isopentenyl transferase, comprising:
  • a protease by deleting, replacing, inserting or adding one or several amino groups to the amino acid in (a) and having the enzymatic activity in a).
  • nucleotide sequence of the gene encoding the aforementioned protease is SEQ ID NO: 2;
  • a recombinant expression vector is provided, and the recombinant expression vector carries the aforementioned gene;
  • the application of the isopentenyl transferase of the present invention is to bind an isopentenyl donor to a cyclic dipeptide compound
  • the isopentenyl donor reagent is dimethylallylpyrophosphate (DMAPP), (geranylpyrophosphate) GPP, (farnesylpyrophosphate) FP or (geranylgeranylpyrophosphate) GGPP.
  • the cyclic dipeptide is cyclo (L-Trp-L-Xaa), where Xaa is Val, Pro, Leu, Ile, Ala, Thr, Gln, Phe or Tyr.
  • the isopentenyl transferase of the present invention generates some drimentine-like precursor compounds through isoprenylation of a cyclic dipeptide, and provides a new strategy for the drug development of diketopiperazines.
  • FIG. 1 Electrophoresis diagram of the construction of the expression vector constructed by the present invention
  • FIG. 1 SDS-PAGE analysis electrophoresis chart of purified isopentenyl transferase DmtC1 in the present invention
  • FIG. 3 High performance liquid chromatography (HPLC) chart of the reaction of DmtC1 with different cyclic dipeptide substrates in the present invention
  • Figure 9 Half inhibition rate of VSV by product 3 in the present invention.
  • a primer pair was designed: P1: 5'-CCGGAATTCACCCGCCAGGAAATGGACG-3 '/ P2: 5'-CCGCTCGAGCGTCGAGTCGGCGGTCAGGG-3', and the T-DNA of Streptomyces youssoufiensis OUC6819 prepared above was diluted 10 times as a template for PCR.
  • the primer pair P1 '/ P2' is used to amplify the functional gene of isopentenyl transferase, and the underlined sites are EcoRI and XhoI, respectively.
  • the constructed recombinant vector was introduced into E. coli BL21 (DE3), and the overnight bacterial solution was inoculated into 1 L of a liquid medium containing 50 ⁇ g mL -1 ampicillin LB, and cultured at 37 ° C to an OD 600 of about 0.8, and 0.05 mM isopropylthio Galactin (IPTG) was further cultured at 16 ° C for 16 hours.
  • the cells were collected by centrifugation, and the cells were re-lysed with a buffer solution (0.05M Tris-HCl, 0.5M NaCl, pH 7.5, containing cOmplete TM protease inhibitor cocktail), and the cells were pulverized, purified by a nickel column, and detected by SDS-PAGE ( Figure 2). ), The higher purity enzyme was concentrated and replaced into a buffer solution (0.025M Tris-HCl, 0.02M NaCl, and 10% glycerol, pH 7.5), and stored at -80 for future use.
  • a buffer solution 0.05M Tris-HCl, 0.5M NaCl, pH 7.5, containing cOmplete TM protease inhibitor cocktail
  • Reaction conditions 30 ° C, 60min. After the reaction, an equal volume of methanol was added to terminate the reaction, and the protein in the reaction system was removed by centrifugation at 17,000xg for 30min. The resulting supernatant was analyzed by HPLC.
  • HPLC detection conditions using reversed-phase YMC-Pack ODS-AQ C18 column (specification: 50mm ⁇ 4.6mm, 5 ⁇ m, ); Column temperature 30 ° C; elution conditions: 0-5min 10% phase A (acetonitrile + 0.1% formic acid) and 90% (ddH 2 O + 0.1% formic acid); 5-15min gradient elution, 10% -50% A phase and 90% -50% B phase; 15.1-25min gradient elution, 80% -100% A phase and 20% -0% B phase; 25-35min isocratic elution, 100% A phase and 0% B Phase; detection wavelength is 300 nm; flow rate is 1 mL min -1 . From the spectrum obtained by HPLC ( Figure 3), a new absorption peak appeared after the reaction, indicating that the enzymatic reaction occurred.
  • Example 3 The effect of DmtC1 on the isoprenyl transfer of different isopentenyl groups
  • Example 4 Vesicular stomatitis virus (VSV) test of product 3 in the above Example 2
  • test sample solution is the product 3 isolated in the above Example 2.
  • DMSO was used to prepare a solution of the required concentration for testing activity.
  • DmtC1 has a broad spectrum of substrates, can use a variety of cyclic dipeptides and isopentenyl as substrates to generate a variety of drimentines precursor compounds, and provides a new drug for the development of diketopiperazines. Strategy.

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Abstract

一种新型的PSL家族异戊烯基转移酶，该酶具有底物广谱性，能以多种环二肽和异戊烯基为底物，产生多种drimentines前体化合物。异戊烯转移酶其氨基酸序列为SEQ ID NO:1。异戊烯转移酶的应用是将异戊烯基供体结合到环二肽类化合物上。异戊烯转移酶通过对环二肽异戊烯化产生一些类drimentine类前体化合物，为二酮哌嗪类化合物的药物研制提供一个新策略。

Description

一种新型异戊烯转移酶及其应用 技术领域
本发明属于基因工程和生物制药技术领域，具体涉及一种新型phytoene-synthase-like(PSL)家族异戊烯转移酶及其应用。
背景技术
异戊烯基广泛存在于许多活性天然产物中，如生物碱、黄酮等化合物。天然产物中异戊烯基的引入不仅极大丰富了化合物结构多样性，而且因其亲脂性的增加而增强了与药物靶点的亲和力及生物利用度，改善药代动力学特性，从而提高了成药性。大量构效关系研究表明，异戊烯基芳香族化合物的生物活性源于芳香类化合物骨架上取代的异戊烯基。由于天然产物结构复杂多样，通过化学法进行异戊烯基化困难，利用底物广谱性异戊烯基转移酶进行异戊烯基化为此提供了新策略。
芳香族异戊烯转移酶可以将异戊烯单元融入含有芳环的化合物，从而形成生物体初级代谢或次级代谢中具有多种生物学功能的活性分子。phytoene-synthase-like(PSL)家族异戊烯转移酶是一类新发现以含有芳环的化合物为底物的异戊烯基转移酶，生物信息学分析显示其属于八氢番茄红素合酶，与典型的芳香族异戊烯转移酶在进化距离上较远。目前唯一报道的PSL家族转移酶是来源于Streptomyces sp.MA37中neocarazostatin生物合成过程中的NzsG，但是它具有极强的底物特异性，只能将dimethylallyl pyrophosphate(DMAPP)转移到precarazostatin。
发明内容
本发明的目的是提供一种新型phytoene-synthase-like(PSL)家族异戊烯基转移酶，具有底物广谱性，能以多种环二肽和异戊烯基为底物，产生多种drimentines前体化合物。
本发明首先提供一种新型异戊烯转移酶，包含有：
a)氨基酸序列为SEQ ID NO:1的蛋白酶；
b)通过对(a)中的氨基酸缺失、取代、插入或添加一个或几个氨基，并 具有a)中酶活性的蛋白酶。
编码上述蛋白酶的基因，其一种核苷酸序列为SEQ ID NO:2；
本发明再一个方面提供一种重组表达载体，所述的重组表达载体中携带有上述的基因；
本发明异戊烯转移酶的应用，是将异戊烯基供体结合到环二肽类化合物上；
所述的异戊烯基供体试剂为dimethylallyl pyrophosphate(DMAPP)、(geranyl pyrophosphate)GPP、(farnesyl pyrophosphate)FP或(geranylgeranyl pyrophosphate)GGPP。
所述的环二肽为cyclo(L-Trp-L-Xaa),其中Xaa为Val、Pro、Leu、Ile、Ala、Thr、Gln、Phe或Tyr。
本发明的异戊烯转移酶通过对环二肽异戊烯化产生一些类drimentine类前体化合物，为二酮哌嗪类化合物的药物研制提供一个新策略。
附图说明
图1：本发明构建的表达载体构建验证电泳图；
图2：本发明中纯化的异戊烯转移酶DmtC1的SDS-PAGE分析电泳图；
图3：本发明中DmtC1与不同环二肽底物反应的高效液相色谱(HPLC)图；
图4：本发明中DmtC1与cWP反应的质谱(MS)谱图；
图5：本发明中DmtC1与cWV反应的质谱(MS)谱图；
图6：本发明中DmtC1与不同异戊烯基反应的高效液相色谱(HPLC)图；
图7：本发明中DmtC1与GPP反应的质谱(MS)谱图；
图8：本发明中DmtC1与GGPP反应的质谱(MS)谱图。
图9：本发明中产物3对VSV的半数抑制率。
具体实施方式
下面结合实施例对本发明做进一步的说明。
实施例1：异戊烯转移酶基因的克隆
1、基因组DNA的提取
将海洋链霉菌Streptomyces youssoufiensis OUC6819接种于TSBY液体培养基中，30℃培养，离心收集菌体，用1mL的STE buffer洗涤；加入500uL STE buffer配制的3～5mg/ml溶菌酶溶液，充分悬浮菌体，于37℃水浴30min， 至细胞成为半透明状；加入250uL 6％的SDS，上下轻轻混匀，继续37℃水浴至澄清；加入1/10体积的3M NaAc(pH＝4.8)后，再加入200uL酚:氯仿:异戊醇(25:24:1；v/v/v)，混合，12000rpm离心；移取上层清液，反复用酚:氯仿:异戊醇抽提，至中间层无蛋白杂质，转移上清，加入等体积的异丙醇，混合直至有白色絮状DNA沉淀析出；挑出絮状沉淀，用70％的乙醇洗涤1次；室温晾干后，用适量的TE溶解基因组DNA，备用。
2、重组载体的构建
设计引物对：P1：5’-CCGGAATTCACCCGCCAGGAAATGGACG-3’/P2：5’–CCGCTCGAGCGTCGAGTCGGCGGTCAGGG-3’，将上述制备的海洋链霉菌Streptomyces youssoufiensis OUC6819的T-DNA稀释10倍作为模板进行PCR。引物对P1’/P2’用来扩增异戊烯转移酶的功能基因，下划线分别为EcoRI和XhoI的酶切位点。
PCR反应体系：
引物对P1和P2各5μl(50pmol)，模板5μl，10×Reaction Buffer 10μl，2.5mM dNTP 10μl，25mM MgCl 2 6μl，pfu DNA Polymerase 1μl(5U/μl)，加ddH 2O至100μl。
PCR条件：
功能基因扩增条件，98℃变性2min；95℃10s，66.3℃15s，72℃10s，25循环；72℃5min。将其克隆到表达载体pET-32a上，构建重组质粒pET-32a-dmtC1，转化大肠杆菌DH5α感受态细胞，挑选阳性克隆，利用EcoRI和XhoI进行酶切验证，结果如图1(panel iii)所示，得到与预期一致大小的目的片段。将验证正确的克隆子进行序列测定，测序结果表明基因的核苷酸序列为SEQ ID NO:2，其编码的蛋白酶的氨基酸序列为SEQ ID NO:1。
3.DmtC1基因在大肠杆菌中的表达与纯化
将构建的重组载体导入大肠杆菌BL21(DE3)中，过夜菌液接种于1L含有50μg mL -1氨苄青霉素LB液体培养基，37℃培养至OD 600约为0.8，加入0.05mM异丙基硫代半乳糖苷(IPTG),于16℃继续培养16小时。离心收集菌体，用缓冲液(0.05M Tris-HCl,0.5M NaCl,pH 7.5,containing cOmplete TM protease inhibitor cocktail)重溶菌体，超生破碎菌体，经镍柱纯化， SDS-PAGE检测(图2)，将纯度较高的酶进行浓缩置换至缓冲液(0.025M Tris-HCl,0.02M NaCl,and 10％glycerol,pH 7.5)中，-80保存备用。
实施例2：DmtC1的体外酶活检测
体外酶活反应体系
50mM Tris-HCl(pH 7.5)缓冲液，2.5mM MgCl 2,10μM DmtC1,1mM环二肽(cWP(1)/cWV(2)),0.2mM FPP
反应条件：30℃,60min,反应结束后加入等体积的甲醇终止反应，17,000xg离心30min除去反应体系中的蛋白。所得上清，进行HPLC检测分析。HPLC检测条件：使用反相YMC-Pack ODS-AQ C18 column(规格：50mm×4.6mm,5μm,
Figure PCTCN2019097372-appb-000001
)；柱温30℃；洗脱条件：0-5min 10％A相(乙腈+0.1％甲酸)和90％(ddH 2O+0.1％甲酸)；5-15min梯度洗脱，10％-50％A相和90％-50％B相；15.1-25min梯度洗脱，80％-100％A相和20％-0％B相；25-35min等度洗脱，100％A相和0％B相；检测波长为300nm；流速为1mL min -1。从HPLC获得的谱图(图3)看，反应后出现了新的吸收峰，说明酶促反应发生。
将获得的产物进行高分辨质谱(HRMS)分析(图4-5)。从MS谱图(图4)上看，panel ii中产物3[M+H] +为488.3274，与FPP-cWP化合物分子量([M+H] +理论值为488.3232)一致；panel iv中产物4[M+H] +为490.3429，与FPP-cWV化合物分子量([M+H] +理论值为490.3389)一致(图5)。
实施例3：DmtC1对不同异戊烯基的转异戊烯作用
以cWP作为底物，将实施例2中酶反应体系中的FPP替换为DMAPP、GPP或GGPP时，终止反应后进行HPLC检测(图6)和HRMS分析(图7-8)，panel iii中产物5的[M+H] +为420.2647，与GPP-cWP化合物分子量([M+H] +理论值为420.2606)一致(图7)；panel v产物6的[M+H] +为556.3896，与GGPP-cWP化合物分子量([M+H] +理论值为556.3858)一致(图8)。从而证明本发明的异戊烯转移酶具有底物广谱性，具有很好的应用前景。
实施例4：上述实施例2中产物3的抗水疱性口炎病毒(Vesicular stomatitis virus,VSV)测试
1、实验样品及实验方法
被测样品溶液为上述实施例2中分离的产物3。用DMSO配制成所需浓度的溶 液，供测试活性。
Vero细胞按5000/孔铺96孔板，用培养基培养12h；用梯度浓度的产物3预处理vero细胞1h，再用VSV-GFP按M.O.I＝0.01感染细胞1h；更换含产物3的培养基继续培养16h；统计平均荧光强度，计算产物3对VSV的半数抑制率(IC 50)。
2、实验结果及结论
产物3对VSV具有显著的抑制作用，其IC 50＝2.66μM(图9)。
工业实用性
申请人在研究海洋链霉菌Streptomyces youssoufiensis OUC6819中异戊烯化二酮哌嗪类化合物drimentines生物合成机制过程中，发现了具有底物广谱性的PSL型异戊烯转移酶DmtC1。体外生化实验研究显示DmtC1具有底物广谱性，能以多种环二肽和异戊烯基为底物，产生多种drimentines前体化合物，为二酮哌嗪类化合物的药物研制提供一个新策略。
Figure PCTCN2019097372-appb-000002
Figure PCTCN2019097372-appb-000003
Figure PCTCN2019097372-appb-000004
Figure PCTCN2019097372-appb-000005

Claims (10)

  1. 一种异戊烯转移酶，其特征在于，所述的异戊烯转移酶包含有：
    a)氨基酸序列为SEQ ID NO:1的蛋白酶；
    b)通过对a)中的氨基酸缺失、取代、插入或添加一个或几个氨基，并具有a)中酶活性的蛋白酶。
  2. 一种基因，其特征在于，所述的基因编码权利要求1所述的异戊烯转移酶。
  3. 如权利要求2所述的基因，其特征在于，所述的基因的核苷酸序列为SEQ ID NO:2。
  4. 一种重组表达载体，其特征在于，所述的重组表达载体中携带有权利要求2所述的基因。
  5. 权利要求1所述的异戊烯转移酶将异戊烯基供体结合到环二肽类化合物上的应用。
  6. 如权利要求5所述的应用，其特征在于，所述的异戊烯基供体试剂为dimethylallyl pyrophosphate、geranyl pyrophosphate、farnesyl pyrophosphate或geranylgeranyl pyrophosphate。
  7. 如权利要求5所述的应用，其特征在于，所述的环二肽为cyclo(L-Trp-L-Xaa),其中Xaa为Val、Pro、Leu、Ile、Ala、Thr、Gln、Phe或Tyr。
  8. 一种将异戊烯基供体结合到环二肽类化合物的方法，其特征在于，所述的方法是使用权利要求1所述的异戊烯转移酶将异戊烯基供体结合到环二肽类化合物上。
  9. 如权利要求8所述的方法，其特征在于，所述的环二肽为cyclo(L-Trp-L-Xaa),其中Xaa为Val、Pro、Leu、Ile、Ala、Thr、Gln、Phe或Tyr。
  10. 如权利要求8所述的方法，其特征在于，的所述的异戊烯基供体试剂为dimethylallyl pyrophosphate、geranyl pyrophosphate、farnesyl pyrophosphate或geranylgeranyl pyrophosphate。
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