WO2021253521A1 - 一种增强微生物固氮能力的人工非编码rna模块 - Google Patents

一种增强微生物固氮能力的人工非编码rna模块 Download PDF

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WO2021253521A1
WO2021253521A1 PCT/CN2020/100412 CN2020100412W WO2021253521A1 WO 2021253521 A1 WO2021253521 A1 WO 2021253521A1 CN 2020100412 W CN2020100412 W CN 2020100412W WO 2021253521 A1 WO2021253521 A1 WO 2021253521A1
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artificial
nitrogen fixation
mrna
aner
module
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林敏�
战嵛华
燕永亮
柯秀彬
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中国农业科学院生物技术研究所
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  • Nitrogen-fixing cells need to express and maintain sufficient nitrogen-fixing gene (nif) mRNAs to ensure high-efficiency nitrogenase activity.
  • Non-coding RNA is a very important type of post-transcriptional regulator. By base pairing with mRNA, it inhibits or activates the expression of target genes at the post-transcriptional level, and then plays an important regulatory role in different metabolic regulation processes of bacteria. It has also been found that multiple ncRNAs may be involved in the regulation of nitrogen-fixing gene expression in nitrogen-fixing bacteria. Among them, the non-coding RNAs NfiR and NfiS cooperate with the nitrogenase genes nifD and nifK mRNA to participate in the regulation of nitrogenase activity.
  • the present invention provides an artificial non-coding RNA module (Ar tificial N itrogenase activity- E nhancing non-coding R NA), named AneR, composed of the following elements:
  • AneR enhances the post-transcriptional stability of nifHDK mRNA by interacting with the nitrogenase-encoding gene nifHDK mRNA.
  • the artificial RNA module AneR of the present invention can significantly improve the nitrogenase activity of various recombinant engineering strains.
  • an artificial RNA function with "degenerate" complementary pairing regions was synthesized by artificial chemical synthesis.
  • Module AneR The expression of AneR is controlled by an artificial promoter that specifically responds to nitrogen fixation signals. It is paired with the bases of the three strains of nitrogenase-encoding gene nifHDK mRNA to cause the inhibited secondary structure to melt, which leads to the inhibition of the nitrogenase gene. Highly expressed, its nucleotide sequence is SEQ ID NO:1.
  • the artificially synthesized AneR fragment was digested with Bam HI and Hind III and inserted into the multiple cloning site of the wide host expression vector pFLA ⁇ 3 to obtain the artificial RNA fusion expression vector pAneR of the present invention ( Figure 1);
  • the expression vectors were transferred into the chassis microbes Pseudomonas stutzeri, Klebsiella pneumoniae, and Azotobacter vinelandii to obtain three recombinant engineering bacteria P. stutzeri (pAneR) , K. pneumoniae (pAneR) and A. vinelandii (pAneR).
  • Microscale Thermophoresis (MST) technology was used to analyze the binding between AneR and nifHDK mRNA, and the results showed that the fitting curves of microthermophoresis between the RNA module AneR and nifH/nifD/nifK mRNA were all typical The "S"-shaped curve indicates that the RNA module AneR and nifH/nifD/nifK mRNA have a good binding trend ( Figure 4).
  • the artificial RNA module AneR of the present invention can significantly improve the nitrogenase activity of various recombinant engineering strains, and significantly enhance the nitrogen-fixing ability of the nitrogen-fixing microorganism chassis.
  • the promoter sequence of the present invention can be used for the effective expression of nitrogen-fixing genes in different bacteria, and therefore can be applied to the construction of various artificial high-efficiency nitrogen-fixing systems.
  • Figure 1 Construction of the artificial RNA module AneR expression vector.
  • the direction of gene transcription in the figure is indicated by arrows, where figure a shows the schematic diagram of the construction of the artificial RNA module AneR expression vector, and the insertion sites are Bam HI and Hind ⁇ ; figure b in the figure shows the PCR verification of the expression vector pAneR.
  • Figure 2 qRT-PCR analysis of the transcription level of the artificial RNA module AneR in the recombinant nitrogen fixation engineering strains P. stutzeri (pAneR), K. pneumoniae (pAneR) and A. vinelandii (pAneR) under nitrogen fixation conditions.
  • SEQ ID NO: 1 The nucleotide sequence of the artificial RNA AneR module.
  • SEQ ID NO: 2 The nucleotide sequence of the gene encoding artificial RNAAneR.
  • the artificial chemical synthesis method was used to obtain the full-length nucleic acid sequence of the artificial RNA module AneR, and the fusion expression vector and the recombinant engineering strain to express the artificial RNA module were successfully constructed.
  • the correct fusion expression vector was verified by PCR sequencing and named pAneR.
  • the three recombinant engineering strains containing pAneR are P. stutzeri (pAneR), K. pneumoniae K. pneumoniae (pAneR) and A. vinelandii (pAneR).
  • Example 2 Expression analysis of the artificial RNA module AneR in the recombinant engineered strain under nitrogen fixation conditions
  • Artificial inducible activation elements can specifically respond to nitrogen fixation signals, thereby initiating the high expression of artificial RNA AneR encoding genes ( Figure 2).
  • the determination of the nitrogenase activity of the recombinant engineering strain adopts the internationally recognized acetylene reduction method.
  • the specific steps are as follows:
  • nitrogenase activity ethylene peak area ⁇ (total gas phase volume of the triangular flask/sampling volume)/(1nmol ethylene standard peak area ⁇ reaction time ⁇ total bacterial protein total).
  • the artificial RNA module AneR induced under nitrogen-fixing conditions can significantly improve the nitrogen-fixing ability of the chassis strains ( Figure 3).
  • Example 4 Identification of the binding ability of artificial RNA AneR and nitrogenase gene nifH/nifD/nifK mRNA
  • the 30bp nifH/nifD/nifK mRNA sequence required for this experiment was separately synthesized by the company (Shanghai Jima Pharmaceutical Technology Co., Ltd.), and 5'FAM fluorescent labeling was carried out as a probe; the full-length 145bp was obtained by in vitro transcription Artificial RNA sequence as a ligand;
  • Kd [A]*[L]/[AL], where [A] is the concentration of free fluorescent molecules, [L] is the concentration of free ligands, and [AL] is the concentration of A and L complexes.
  • the artificial RNA AneR can interact with the nitrogenase gene nifH/nifD/nifK mRNA through base complementary pairing ( Figure 4).
  • the culture solution was shaken and cultured at 30°C for 5 hours;
  • RNAlater (2 times the volume of rifampicin) to the bacterial cells from which the supernatant is removed, suspend the bacterial cells, and treat them at room temperature for 5 minutes, then quickly centrifuge, remove the supernatant, and freeze with liquid nitrogen;
  • nifH/nifD/nifK mRNA in the chassis strain P.stutzeri A1501 is about 20 minutes, while the half-life of nifH/nifD/nifK mRNA in the recombinant strain P.stutzeri (pAneR) is about 25 minutes.
  • the artificial RNA module AneR induced under nitrogen fixation conditions can enhance the stability of nitrogenase mRNA ( Figure 5).

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Abstract

利用合成生物学方法创建的人工非编码RNA模块及其在人工固氮体系构建中的用途。所述RNA模块通过与固氮酶编码基因nifHDK mRNA的相互作用，增强nifHDK mRNA转录后的稳定性，进而提高了底盘微生物的固氮能力。构建了人工RNA模块的融合表达载体，并转入到不同的底盘固氮微生物中。实验证实，在固氮条件下，人工RNA模块能够显著提高重组工程菌株固氮酶活性。

Description

一种增强微生物固氮能力的人工非编码RNA模块 技术领域：
本发明涉及生物技术领域，具体涉及一种增强微生物固氮能力的人工非编码RNA模块及其在固氮合成生物学中的应用。
背景技术：
生物固氮是固氮微生物特有的一种生理功能，这种功能是在固氮酶的催化作用下进行的，受胞内能源供应和环境胁迫因子影响较大。为适应环境变化，固氮微生物在进化过程中形成了一套复杂的调控系统，固氮细胞需要表达和维持足够的固氮基因(nif)mRNAs分子，才能确保高效固氮酶活。
天然固氮体系由于受环境影响较大，导致固氮效率低下，从而极大地限制了其在农业生产中的应用。因此利用合成生物学方法，人工设计标准化、智能化的信号响应元件及功能模块，构建高效固氮基因回路，在微生物底盘中进行适配机制研究，创制新型人工固氮体系，是提高生物固氮效率，实现其在农业广泛应用的新策略和新途径。
非编码RNA(Non-coding RNA,ncRNA)是一类非常重要的转录后调节子。其通过与mRNA碱基配对，在转录后水平抑制或是激活靶基因的表达，进而在细菌不同的代谢调控过程中发挥重要的调节作用。在固氮菌中也发现了多个ncRNAs可能参与固氮基因的表达调控，其中非编码RNA NfiR和NfiS分别通过与固氮酶基因nifD和nifK mRNA的相互作用协同参与固氮酶活的调控。
因此，人工设计ncRNAs，利用其参与固氮酶调控的作用特性可以构建人工高效固氮体系。
发明内容：
本发明的目的是设计与合成一种人工非编码RNA模块，用于各种人工高效固氮体系构建。
本发明提供了一种人工非编码RNA模块( Artificial  Nitrogenase activity- Enhancing non-coding  RNA)，命名为AneR，由以下元件构成：
(1)含有与固氮酶编码基因nifHDK mRNA互补配对区的人工RNA编码序列，其核苷酸序列由SEQ ID NO:2所示
(3)启动所述人工RNA编码基因转录的σ 54依赖型人工启动子元件，其核苷酸序 列由SEQ ID NO:3所示。
本发明的人工非编码RNA模块AneR提高底盘微生物的固氮能力的原理是：AneR通过与固氮酶编码基因nifHDK mRNA的相互作用，增强nifHDK mRNA转录后的稳定性。
本发明构建了该人工RNA模块的表达载体pAneR，其表达受固氮条件诱导表达的人工启动子控制，将该融合载体分别转至固氮施氏假单胞菌、肺炎克氏杆菌及棕色固氮菌3株固氮微生物底盘中，获得了3个具有新型人工固氮回路的重组工程菌株。
实验证实，在固氮条件下，本发明的人工RNA模块AneR能够显著提高多种重组工程菌株固氮酶活性。
本发明是通过以下具体工作得到上述人工RNA功能元件模块，并证实其功能：
1、人工RNA模块AneR的设计与合成
根据固氮施氏假单胞菌、肺炎克氏杆菌以及棕色固氮菌中固氮酶编码基因nifHDK mRNA的序列保守性，用人工化学合成的方法合成一个具有“简并性”互补配对区的人工RNA功能模块AneR。AneR的表达受一个由特异响应固氮信号的人工启动子控制，其通过与三株菌固氮酶编码基因nifHDK mRNA的碱基互补配对，引起受抑制的二级结构解链，从而导致固氮酶基因的高效表达，其核苷酸序列是SEQ ID NO：1。
2、构建人工RNA模块AneR的融合表达载体，将其转入三个不同的固氮微生物底盘中，获得三个重组固氮工程菌株
(1)构建AneR融合表达载体
将人工合成的AneR片段进行Bam HI和Hind III双酶切，插入到广宿主表达载体pFLAα3的多克隆位点处，获得本发明人工RNA的融合表达载体pAneR(图1)；
(2)获得三种重组固氮工程菌株
将表达载体分别转入底盘微生物固氮施氏假单胞菌(Pseudomonas stutzeri)、肺炎克氏杆菌(Klebsiella pneumoniae)及棕色固氮菌(Azotobacter vinelandii)中，获得三种重组工程菌P.stutzeri(pAneR)、K.pneumoniae(pAneR)和A.vinelandii(pAneR)。
3、重组固氮工程菌株的固氮能力分析
(1)人工RNA模块AneR对固氮胁迫信号的响应分析
利用qRT-PCR技术对固氮条件下三株重组菌株中的人工RNA模块的表达量进行分析，结果显示：与非固氮条件相比，在固氮条件下三株重组菌株中AneR的转录水平显著提高了1.5倍以上(图2)。
(2)重组固氮工程菌株固氮酶活性测定
利用乙炔还原法比较固氮条件下底盘菌株与重组菌株的固氮酶活性，结果显示：与底 盘菌株相比，三株重组菌株固氮酶活性显著提高了20％左右(图3)。
4、人工RNA模块AneR与固氮酶编码基因nifHDK mRNA结合能力分析
利用微量热泳动(Microscale Thermophoresis,MST)技术分析AneR与nifHDK mRNA之间的结合情况，结果显示：RNA模块AneR分别与nifH/nifD/nifK mRNA之间的微量热泳动拟合曲线均为典型的“S”型曲线，说明RNA模块AneR与nifH/nifD/nifK mRNA之间均有很好的结合趋势(图4)。
5、人工RNA功能模块AneR对固氮酶基因nifHDK mRNA的稳定性分析
利用qRT-PCR技术分析固氮条件下A1501和P.stutzeri(pAneR)中nifHDK mRNA的半衰期，结果显示：重组菌中的nifHDK mRNA的半衰期明显变长，其中重组菌中的nifHDK mRNA的半衰期约为25min，而野生型中的约为20min(图5)。
本发明的有益效果
实验证实，在固氮条件下，本发明的人工RNA模块AneR能够显著提高多种重组工程菌株固氮酶活性，明显增强固氮微生物底盘的固氮能力。
本发明的启动子序列可以用于不同细菌中固氮基因的有效表达，因此可应用于各种人工高效固氮体系的构建。
附图说明
图1：人工RNA模块AneR表达载体的构建。图中基因转录方向用箭头表示，其中a图表示人工RNA模块AneR表达载体的构建示意图，插入位点为Bam HI和HindШ；图中b图表示表达载体pAneR的PCR验证。
图2：qRT-PCR分析固氮条件下重组固氮工程菌株P.stutzeri(pAneR)、K.pneumoniae(pAneR)和A.vinelandii(pAneR)中人工RNA模块AneR的转录水平。
图3：底盘菌株和重组工程菌株P.stutzeri(pAneR)、K.pneumoniae(pAneR)和A.vinelandii(pAneR)固氮酶活性测定。
图4：人工RNA模块AneR与固氮酶编码基因nifH/nifD/nifK mRNA结合能力测定。
图5：底盘微生物P.stutzeri A1501和重组菌株P.stutzeri(pAneR)中固氮酶基因nifHDK mRNA半衰期测定
序列信息
SEQ ID NO:1人工RNA AneR模块的核苷酸序列。
SEQ ID NO:2人工RNAAneR编码基因的核苷酸序列。
SEQ ID NO:3σ 54依赖型人工启动子的核苷酸序列。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明本发明的方法，而不用于限制本发明的范围。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1构建AneR融合表达载体
(一)实验方法：
首先通过人工化学合成的方法合成一个全长为505bp的人工RNA模块AneR，其表达受大小为360bp的人工启动元件控制，然后分别将人工RNA模块AneR和表达载体pFLAα3进行Bam HI和Hind III双酶切，利用T4DNA连接酶将酶切回收的AneR片段插入到pFLAα3的多克隆位点处，最后经PCR测序验证，获得了AneR融合表达载体pAneR。将表达载体通过三亲结合或电激的方式分别转入三种不同的固氮微生物底盘(固氮施氏假单胞菌P.stutzeri、肺炎克氏杆菌K.pneumoniae及棕色固氮菌A.vinelandii)中，获得三个重组固氮工程菌株。
(二)实验结果：
利用人工化学合成的方法获得了人工RNA模块AneR全长核酸序列，成功构建了表达人工RNA模块的融合表达载体和重组工程菌株。经PCR测序验证融合表达载体正确，命名为pAneR。含有pAneR的三株重组工程菌株为固氮施氏假单胞菌P.stutzeri(pAneR)、肺炎克氏杆菌K.pneumoniae(pAneR)和棕色固氮菌A.vinelandii(pAneR)。
(三)实验结论：
完成人工RNA模块AneR融合表达载体以及重组固氮工程菌株的构建。
实施例2固氮条件下重组工程菌株中人工RNA模块AneR的表达分析
(一)实验方法
1.将重组菌株P.stutzeri(pAneR)、K.pneumoniae(pAneR)和A.vinelandii(pAneR)在LB液体培养基中活化，30℃过夜培养；
2.次日4000rpm/10min离心菌体，用生理盐水洗涤菌体两次；
3.用生理盐水悬浮菌体，调OD 600≈1.0；
4.分别将菌体在正常条件和固氮条件下培养，调OD 600≈0.5；
5.培养液30℃震荡培养0.5h后，8000rpm离心5min收集菌体；
6.采用Promega大量提取RNA试剂盒Z3741提取细菌总RNA，将使用量相同的样品RNA进行单链DNA(cDNA)反转；
7.通过qRT-PCR方法对固氮条件下人工RNA模块AneR的表达水平进行了分析。
(二)实验结果：
发现固氮条件下三株重组菌株中aneR的转录水平显著提高，相对非固氮条件提高了1.5倍以上。
(三)实验结论：
人工诱导型启动元件能够特异的响应固氮信号，从而启动人工RNA AneR编码基因的高表达(图2)。
实施例3重组固氮工程菌株的固氮酶活测定
(一)实验方法
重组工程菌株固氮酶活的测定采用了国际上普遍公认的乙炔还原法，具体步骤如下：
1.将底盘菌株P.stutzeri、K.pneumoniae及A.vinelandii和重组菌株P.stutzeri(pAneR)、K.pneumoniae(pAneR)及A.vinelandii(pAneR)在LB液体培养基中活化，30℃过夜培养；
2.次日4000rpm/10min离心菌体，用生理盐水洗涤菌体两次；
3.用生理盐水悬浮菌体，调OD 600≈1.0，将菌体分别在以下条件下培养：
P.stutzeri和重组菌P.stutzeri(pAneR)
(1)将菌悬液分别转接至装有K培养基(无N)磨口三角瓶中，调OD 600≈0.1，塞上橡胶塞；
(2)充氩气排空气三分钟，然后利用微量取样器向每个瓶中注入1％的氧气，和10％的乙炔。30℃，200rpm培养；
A.vinelandii和重组菌A.vinelandii(pAneR)
(1)将菌悬液分别转接至装有Buik's培养基(无N)磨口三角瓶中，调OD 600≈0.1，塞上橡胶塞；
(2)利用微量取样器向每个瓶中注入10％的乙炔。30℃，200rpm培养；
K.pneumoniae和重组菌K.pneumoniae(pAneR)
(1)将菌悬液分别转接至装有含有蔗糖的基本培养基(无N)和氦气的磨口三角瓶中，调OD 600≈0.1，塞上橡胶塞；
(2)利用微量取样器向每个瓶中注入10％的乙炔。30℃，200rpm培养；
4.培养四小时后取样。利用微量取样器吸取0.25mL的三角瓶气体。注入气相色谱仪中，记录乙烯和乙炔的峰面积。每小时取样进行测量；
5.利用考马斯亮蓝法测定三角瓶中菌液的全蛋白含量；
6.利用公式固氮酶活＝乙烯峰面积×(三角瓶的气相总体积/取样体积)/(1nmol乙烯标准峰面积×反应时间×菌体全蛋白总量)。
(二)实验结果：
与底盘固氮菌相比，三株重组固氮工程菌株的固氮酶活性明显提高。
(三)实验结论：
固氮条件下诱导表达的人工RNA模块AneR能够显著提高底盘菌株的固氮能力(图3)。实施例4人工RNA AneR与固氮酶基因nifH/nifD/nifK mRNA结合能力鉴定
(一)实验方法
1.RNA的合成与标记
由公司分别合成(上海吉玛制药技术有限公司)本实验所需的长度为30bp的nifH/nifD/nifK mRNA序列，进行5'FAM荧光标记作为探针；通过体外转录的方法获得全长145bp的人工RNA序列，作为配体；
2.探针与配体的混合反应
含有200nM标记探针和增加非标记竞争物浓度的样品(从5nm到150μM)被分别加入到16个标准处理的毛细管中，静置5min
3.微量热涌动测量和数据分析
利用NT.115仪器(NanoTemper Technologies GmbH)对RNA模块与nifH/nifD/nifK mRNA之间的结合能力进行分析并计算出解离常数Kd。Kd＝[A]*[L]/[AL]，其中[A]是自由荧光分子的浓度，[L]是自由配体的浓度，[AL]是A和L复合物的浓度。
(二)实验结果：
人工RNA AneR分别与nifH/nifD/nifK mRNA之间的微量热泳动拟合曲线均为典型的“S”型曲线，说明RNA AneR与nifH/nifD/nifK mRNA之间均有很好的结合趋势(图4)。
(三)实验结论：
人工RNA AneR能够通过碱基互补配对的方式分别与固氮酶基因nifH/nifD/nifK mRNA相互作用(图4)。
实施例5重组固氮工程菌株中固氮酶基因nifHDK mRNA半衰期测定
(一)实验方法
1.将P.stutzeri A1501和重组菌株P.stutzeri(pAneR)LB液体培养基中活化，30℃过夜培养；
2.次日4000rpm/10min离心菌体，用生理盐水洗涤菌体两次；
3.用生理盐水悬浮菌体，调OD 600≈1.0；
4.将菌悬液分别转接至装有K培养基和K培养基(无N)磨口三角瓶中，调OD 600≈0.5，塞上橡胶塞；
5.充氩气排空气三分钟，然后利用微量取样器向每个瓶中注入1％的氧气，和10％的乙炔；
6.培养液30℃震荡培养5h；
7.菌液中加入40mg/mL的利福平母液，混匀。在菌液分别处理0,5,10,15,20,25和30min后，吸取2mL菌液于1.5mL EP管中，12000rpm，2min迅速离心去除上清；
8.向去除上清的菌体中加入400μL的RNAlater(2倍利福平体积)，悬浮上述菌体，室温条件下处理5min后，迅速离心，去除上清，液氮速冻；
9.提取样品RNA，反转录合成cDNA，qRT-PCR检测nifHDK mRNA的半衰期。
(二)实验结果：
底盘菌株P.stutzeri A1501中的nifH/nifD/nifK mRNA的半衰期约为20min，而重组菌株P.stutzeri(pAneR)中的nifH/nifD/nifK mRNA的半衰期约为25min。
(三)实验结论：
固氮条件下诱导表达的人工RNA模块AneR能够增强固氮酶mRNA的稳定性(图5)。

Claims (8)

  1. 核苷酸序列如SEQ ID NO：2所示的人工RNA编码序列。
  2. 权利要求1所述的人工RNA编码序列与固氮酶基因nifHDK mRNA相互作用的用途。
  3. 权利要求1所述的人工RNA编码序列在构建人工固氮体系中的应用。
  4. 含权利要求1所述的人工RNA编码序列的质粒。
  5. 含权利要求1所述的人工RNA的重组工程菌株。
  6. 权利要求4所述的重组工程菌株在构建人工固氮体系中的应用。
  7. 一种人工RNA模块，含核苷酸序列如SEQ ID NO：2所示的人工RNA编码序列。
  8. 含权利要求6所述人工RNA模块的融合表达载体在构建人工固氮体系中的应用。
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