CN111926048B - 烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用 - Google Patents

烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用 Download PDF

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CN111926048B
CN111926048B CN202010698336.5A CN202010698336A CN111926048B CN 111926048 B CN111926048 B CN 111926048B CN 202010698336 A CN202010698336 A CN 202010698336A CN 111926048 B CN111926048 B CN 111926048B
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folic acid
pseudomonas aeruginosa
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allyl sulfide
biosynthesis
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李文茹
曾桃花
张志清
谢小保
李素娟
施庆珊
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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Abstract

本发明公开了烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用。本发明发现DAS不会抑制铜绿假单胞菌PAO1菌株的生长，但能够使该菌叶酸合成通路中的关键基因表达上调，提高该菌的叶酸生物合成水平。因此烯丙基硫醚可应用于细菌发酵生产叶酸工业中，提高叶酸的发酵产量。

Description

烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的 应用
技术领域：
本发明属于细菌发酵技术领域，具体涉及烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用。
背景技术：
大蒜是常见的食物，其用作民间医药的历史已有几千年。现代科学研究证明，大蒜精油中含硫化合物具有较强的抗菌消炎作用，对多种细菌、真菌和病毒均有抑制作用。烯丙基硫醚(Diallyl sulfide，DAS)是大蒜精油中一种含硫丰富的化合物。叶酸，即维生素B9，它是一种水溶性的B族维生素。叶酸在绿叶蔬菜、酵母和动物肝脏中含量丰富，是人体必需的一种非常重要的维生素，但人类自身不能合成叶酸，必须依靠食物供给。人体缺乏叶酸会引起红细胞异常，贫血以及白细胞的减少。叶酸也是胎儿生长发育不可缺少的营养元素，孕妇缺乏叶酸可引起胎儿发育缺陷和畸形。
发明内容：
本发明的目的是提供一种烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用。
本发明通过实验表明，烯丙基硫醚(DAS)能够提高了铜绿假单胞菌培养液中叶酸的产量。
因此，本发明的目的是提供烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用。
本发明的第二个目的是提供一种促进铜绿假单胞菌叶酸生物合成制剂，含有烯丙基硫醚作为活性成分。
所述的铜绿假单胞菌优选为铜绿假单胞菌(Pseudomonas aeruginosa)PAO1。
本发明发现DAS能够提高了铜绿假单胞菌培养液中叶酸的产量，因此可以用于制备促进铜绿假单胞菌叶酸生物合成制剂中应用。
附图说明：
图1是DAS作用下铜绿假单胞菌PAO1的生长曲线；
图2是DAS作用下铜绿假单胞菌PAO1的转录组KEGG富集分析的叶酸合成通路图。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
铜绿假单胞菌(Pseudomonas aeruginosa)PAO1悬液的制备：将指数生长期的铜绿假单胞菌PAO1培养液取样，离心，用PBS缓冲液洗涤一次，重悬于PBS中，并将菌浓度稀释至108CFU/mL，得到铜绿假单胞菌PAO1菌悬液。
1.烯丙基硫醚对铜绿假单胞菌生长的影响
分别向试管中加入LB培养基、DAS，并接入指数生长期的铜绿假单胞菌PAO1菌悬液，总体积均为10mL，使铜绿假单胞菌PAO1的菌浓度均为106CFU/mL，DAS的浓度分别为0(对照)、0.16μl/mL、0.31μl/mL、0.63μl/mL、1.25μl/mL、2.5μl/mL和5.0μl/mL。将几个实验组分别取样加入到自动生长曲线测定仪(Bioscreen C)专用的蜂窝培养板中，每孔加入350μL的培养液，每个实验组三个平行。将蜂窝培养板置于自动生长曲线测定仪中，37℃振荡培养3天，每小时测定一次OD600。以OD600为纵坐标，培养时间为横坐标，绘制DAS作用下PAO1的生长曲线，以此研究DAS对PAO1生长的影响。
2.烯丙基硫醚对铜绿假单胞菌的叶酸合成通路中关键基因表达量的影响
向50mL无菌LB液体培养基中加入处于对数生长期的PAO1菌悬液，使菌液终浓度为106CFU/mL。加入DAS，使得终浓度为0(对照组三个生物学重复分别命名为A1、A2、A3)和0.63μl/mL(实验组三个生物学重复分别命名为B1、B2、B3)；将各组置于37℃、180rpm条件下培养5h；离心并收集菌体，-80℃速冻后备用。
使用Trizol(Thermo公司)的试剂盒提菌体总RNA。提取后用超微量分光光度计(Implen,Munich,德国)检测RNA的纯度。每份RNA样品的A260/A280值应该在1.8～2.0之间。转录组测序在华大基因有限公司(深圳，中国)完成。参考基因组和基因模型注释文件直接从NCBI基因组网站下载。使用bowtie2-2.2.3构建参考基因组索引并将clean reads比对到参考基因组。Rockhopper被用来识别新基因、操纵子和转录起始位点。然后，利用时延神经网络提取转录起始位点上游700个碱基对，进行启动子预测。用DESeq R包(1.18.0)进行组间差异表达分析，通过使用Benjamini和Hochberg的方法控制伪发现率，调整得到的p值。经DESeq鉴定，基因的校正p值<0.05，则为差异表达。《京都基因与基因组百科全书》(KEGG)数据库是一种从分子水平信息了解各种生物系统功能和属性的资源库，特别是指通过基因组测序等高通量实验技术生成的大规模分子数据集。用KOBAS软件进行差异表达基因KEGG通路富集分析。
实验结果：
1.烯丙基硫醚不影响铜绿假单胞菌的生长
不同浓度的DAS作用下，铜绿假单胞菌PAO1的生长曲线见图1。从图中可以看出，对照组中铜绿假单胞菌的生长曲线包含了生长延滞期、指数生长期、稳定期和衰亡期，不同浓度梯度的DAS暴露下，铜绿假单胞菌的生长曲线与对照组相比，没有显著的差异和变化，与对照组的生长趋势一致。因此，DAS不影响铜绿假单胞菌的生长。
2.烯丙基硫醚增强了铜绿假单胞菌叶酸生物合成通路中关键基因的表达水平
DAS处理5h后，铜绿假单胞菌的转录组差异表达基因KEGG富集分析中富集到了叶酸生物合成通路(One carbon pool by folate)，该通路中的详细差异表达基因见图2。图2的KEGG富集叶酸生物合成通路图中，1.5.1.3、2.1.2.3、3.5.1.10、2.1.2.9、2.1.2.2、2.1.1.13、2.1.1.45、3.5.4.9、6.3.3.2、1.5.1.5、2.1.2.10、2.1.2.1的RNA为表达水平显著上调，其他节点的RNA为表达水平不变。从图2中可以看出，参与叶酸生物合成的关键基因中大部分都发生了表达上调。实验结果表明，DAS能够增强铜绿假单胞菌的叶酸生物合成能力，提高该菌的叶酸生物合成水平。
综上的实验结果表明，DAS不会影响铜绿假单胞菌的生长，但是会提高该菌的叶酸生物合成水平。因此，DAS可以应用到细菌发酵生产叶酸过程中，用以提高细菌发酵生产叶酸的产量。

Claims (1)

1.烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成关键基因表达上调制剂中的应用，所述的铜绿假单胞菌为铜绿假单胞菌（Pseudomonas aeruginosa）PAO1；
所述的关键基因的数字代码、Gene ID和基因别名如下所示：
。
CN202010698336.5A 2020-07-20 2020-07-20 烯丙基硫醚在制备促进铜绿假单胞菌叶酸生物合成制剂中的应用 Active CN111926048B (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102675168A (zh) * 2011-03-07 2012-09-19 同济大学 一种烷烃类手性烯丙基硫醚化合物及其制备方法
CN105768092A (zh) * 2016-04-08 2016-07-20 厦门元之道生物科技有限公司 一种具有抗菌活性的综合发酵液及其用途
CN109498609A (zh) * 2018-10-30 2019-03-22 广东省微生物研究所（广东省微生物分析检测中心） 烯丙基硫醚在制备铜绿假单胞菌群体感应系统淬灭剂中的应用

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MX2020005875A (es) * 2017-12-05 2020-10-08 Bioplx Inc Métodos y composiciones para prevenir la infección microbiana.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102675168A (zh) * 2011-03-07 2012-09-19 同济大学 一种烷烃类手性烯丙基硫醚化合物及其制备方法
CN105768092A (zh) * 2016-04-08 2016-07-20 厦门元之道生物科技有限公司 一种具有抗菌活性的综合发酵液及其用途
CN109498609A (zh) * 2018-10-30 2019-03-22 广东省微生物研究所（广东省微生物分析检测中心） 烯丙基硫醚在制备铜绿假单胞菌群体感应系统淬灭剂中的应用

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