CN118165906A - A recombinant bacterium for directly utilizing waste carbon dioxide to produce 3-hydroxypropionic acid and its construction method and application - Google Patents

Abstract

The invention provides a recombinant bacterium for producing 3-hydroxy propionic acid by directly utilizing waste carbon dioxide, a construction method and application thereof, and discloses nucleotide sequences of a coding malonyl-CoA reductase gene MCR and a coding malonate semialdehyde reductase gene MSR after codon optimization aiming at thermophilic cyanobacteria E542, belonging to the technical field of genetic engineering. Aiming at the biosynthesis of 3-hydroxy propionic acid, the invention provides a recombinant thermophilic cyanobacterium directly utilizing waste carbon dioxide as a raw material to carry out the biosynthesis of 3-hydroxy propionic acid, wherein the strain is obtained by introducing malonyl-CoA reductase gene MCR and malonate semialdehyde reductase gene MSR into thermophilic cyanobacterium E542 through free shuttle plasmids. The recombinant thermophilic cyanobacteria prepared by the method realizes the biosynthesis of the 3-hydroxy propionic acid by directly utilizing the waste carbon dioxide as a raw material, is safe to produce, environment-friendly, low in cost and sustainable, and provides a new thought for further green renewable production of the 3-hydroxy propionic acid.

Description

Translated fromChinese

一种直接利用废弃二氧化碳产3-羟基丙酸的重组菌及其构建方法与应用A recombinant bacterium that directly utilizes waste carbon dioxide to produce 3-hydroxypropionic acid and its construction method and application

技术领域Technical Field

本发明涉及一种直接利用废弃二氧化碳产3-羟基丙酸的重组菌及其构建方法与应用，并公开了其生物合成关键基因的核苷酸序列，属于基因工程技术领域。The invention relates to a recombinant bacterium for directly utilizing waste carbon dioxide to produce 3-hydroxypropionic acid, a construction method and application thereof, and discloses the nucleotide sequence of a key gene for its biosynthesis, belonging to the technical field of genetic engineering.

背景技术Background technique

3-羟基丙酸是一种多功能平台化学品，是1,3-丙二醇、3- 羟基丙醛、丙烯酸和丙二酸等多种化工原料的重要前体，并可用于合成多种生物可降解聚合材料。3-羟基丙酸分子中的羟基和羧基可以聚合形成聚 (3-羟基丙酸酯)[P(3HP]，[P(3HP)]也是一种具有生物兼容性、高机械强度和拉伸强度的可生物降解材料，应用广泛。此外，3-羟基丙酸还可以用于合成医用骨骼和塑料产品，在医药和化工领域具有重要的应用前景。3-Hydroxypropionic acid is a multifunctional platform chemical. It is an important precursor of many chemical raw materials such as 1,3-propylene glycol, 3-hydroxypropionaldehyde, acrylic acid and malonic acid, and can be used to synthesize a variety of biodegradable polymers. The hydroxyl and carboxyl groups in the 3-hydroxypropionic acid molecule can be polymerized to form poly (3-hydroxypropionate) [P(3HP], [P(3HP)] is also a biodegradable material with biocompatibility, high mechanical strength and tensile strength, and is widely used. In addition, 3-hydroxypropionic acid can also be used to synthesize medical bones and plastic products, and has important application prospects in the fields of medicine and chemical industry.

目前，3-羟基丙酸的生产以化学合成为主，主要以丙烯酸、乙烯酮、3-羟基丙腈、醋酸乙烯酯和1, 3-丙二醇为前体物质，通过一系列化工路线进行转化。化学法合成 3-羟基丙酸的操作相对简单，反应条件温和，且产率较为稳定，但化学合成法也具有环境污染大、能耗高、产物分离过程复杂、副产物繁多等缺点，限制了其进一步产业化应用。生物法合成3-羟基丙酸则可以有效避免这些不利因素。生物转化法合成3-羟基丙酸的相关研究起步于近二十年，主要是利用重组大肠杆菌和酿酒酵母等异养生物以甘油或葡萄糖等有机化合物为底物进行微生物转化，研究主要集中于工程菌的构建优化和发酵工艺的改进，以期有效提高3-羟基丙酸产量。At present, the production of 3-hydroxypropionic acid is mainly based on chemical synthesis, mainly using acrylic acid, vinyl ketone, 3-hydroxypropionitrile, vinyl acetate and 1, 3-propylene glycol as precursors, and converting them through a series of chemical routes. The operation of chemical synthesis of 3-hydroxypropionic acid is relatively simple, the reaction conditions are mild, and the yield is relatively stable, but the chemical synthesis method also has the disadvantages of large environmental pollution, high energy consumption, complex product separation process, and numerous by-products, which limits its further industrial application. The biological synthesis of 3-hydroxypropionic acid can effectively avoid these unfavorable factors. The relevant research on the synthesis of 3-hydroxypropionic acid by biotransformation started in the past two decades, mainly using heterotrophic organisms such as recombinant Escherichia coli and Saccharomyces cerevisiae to carry out microbial transformation with organic compounds such as glycerol or glucose as substrates. The research mainly focuses on the construction and optimization of engineering bacteria and the improvement of fermentation processes, in order to effectively increase the yield of 3-hydroxypropionic acid.

然而截止目前为止，尚未有通过嗜热自养蓝细菌进行3-羟基丙酸生产的相关报道。该重组菌株不仅能直接进行废气二氧化碳的资源化，而且还可以利用废气二氧化碳所携带的余热对嗜热菌株进行培养，实现真正意义的节能减排。与此同时，在高温环境下进行二氧化碳的直接利用，还可以降低废气二氧化碳的冷却成本。此外，对于工程菌株发酵培养而言，耐高温的嗜热菌株更不容易被其他杂质污染，有利于进一步降低发酵生产成本。因此，本发明利用本研究团队前期自主分离的嗜热蓝细菌E542作为宿主菌株，将编码丙二酰-CoA还原酶基因MCR和编码丙二酸半醛还原酶基因MSR整合到穿梭的游离质粒pRS416-kana上，并将重组质粒导入构建重组嗜热蓝细菌E542，通过发酵培养检测在高温环境下重组嗜热蓝细菌E542生物合成3-羟基丙酸的水平。However, up to now, there is no relevant report on the production of 3-hydroxypropionic acid by thermophilic autotrophic cyanobacteria. The recombinant strain can not only directly carry out the resource of waste gas carbon dioxide, but also can utilize the waste heat carried by waste gas carbon dioxide to cultivate thermophilic strains, realize real energy saving and emission reduction. At the same time, the direct utilization of carbon dioxide under high temperature environment can also reduce the cooling cost of waste gas carbon dioxide. In addition, for engineering strain fermentation culture, thermophilic strains resistant to high temperature are less likely to be contaminated by other impurities, which is conducive to further reducing the fermentation production cost. Therefore, the present invention utilizes the thermophilic cyanobacteria E542 that this research team separated autonomously in the early stage as the host strain, and the encoding malonyl-CoA reductase gene MCR and the encoding malonate semialdehyde reductase gene MSR are integrated into the shuttle free plasmid pRS416-kana, and the recombinant plasmid is imported to construct the recombinant thermophilic cyanobacteria E542, and the level of the biosynthesis of 3-hydroxypropionic acid of the recombinant thermophilic cyanobacteria E542 under high temperature environment is detected by fermentation culture.

发明内容Summary of the invention

本发明目的在于提供一种直接利用废弃二氧化碳产3-羟基丙酸的重组菌及其构建方法与应用，并公开了其生物合成关键基因的核苷酸序列，具体技术方案如下：The present invention aims to provide a recombinant bacterium that directly utilizes waste carbon dioxide to produce 3-hydroxypropionic acid, a construction method and application thereof, and discloses the nucleotide sequence of its key biosynthetic gene. The specific technical scheme is as follows:

一种直接利用废气二氧化碳产3-羟基丙酸的重组菌，所述重组菌是将编码丙二酰-CoA还原酶基因MCR和编码丙二酸半醛还原酶基因MSR通过穿梭质粒pRS416-kana导入嗜热蓝细菌E542中获得的；基于嗜热蓝细菌E542对所述编码丙二酰-CoA还原酶基因MCR和编码丙二酸半醛还原酶基因MSR进行了密码子偏好性优化，所述MCR经过密码子优化后的核苷酸序列如SEQ ID NO.1所示；所述MSR经过密码子优化后的核苷酸序列如SEQ ID NO.2所示。A recombinant bacterium for directly utilizing waste gas carbon dioxide to produce 3-hydroxypropionic acid. The recombinant bacterium is obtained by introducing a gene MCR encoding malonyl-CoA reductase and a gene MSR encoding malonate semialdehyde reductase into thermophilic cyanobacteria E542 via a shuttle plasmid pRS416-kana; based on thermophilic cyanobacteria E542, the gene MCR encoding malonyl-CoA reductase and the gene MSR encoding malonate semialdehyde reductase are codon-optimized, and the nucleotide sequence of the MCR after codon optimization is shown in SEQ ID NO.1; the nucleotide sequence of the MSR after codon optimization is shown in SEQ ID NO.2.

在本发明的一个实施例中，所述编码丙二酰-CoA还原酶基因MCR和编码丙二酸半醛还原酶基因MSR均来源于嗜热金属球菌Metallosphaera sedula。In one embodiment of the present invention, the gene MCR encoding malonyl-CoA reductase and the gene MSR encoding malonate semialdehyde reductase are both derived fromMetallosphaera sedula .

在本发明的一个实施例中，丙二酰-CoA还原酶在NCBI的蛋白质编号是A4YEN2.1；丙二酸半醛还原酶在NCBI的蛋白质编号是ABP96133.1。In one embodiment of the present invention, the protein number of malonyl-CoA reductase in NCBI is A4YEN2.1; the protein number of malonate semialdehyde reductase in NCBI is ABP96133.1.

本发明还提供了上述重组菌的构建方法，步骤如下：The present invention also provides a method for constructing the above-mentioned recombinant bacteria, the steps of which are as follows:

1）分别合成编码丙二酰-CoA还原酶基因MCR和丙二酸半醛还原酶基因MSR。1) The genes encoding malonyl-CoA reductase (MCR) and malonate semialdehyde reductase (MSR) were synthesized respectively.

选择启动子PcpcB1₇₉₄₂，其核苷酸序列SEQ ID NO.3；选择终止子TrrnBT1，其核苷酸序列SEQ ID NO.4；通过PCR融合获得丙二酰-CoA还原酶基因MCR的表达框，其核苷酸序列SEQ ID NO.5。The promoter PcpcB1₇₉₄₂ was selected, and its nucleotide sequence was SEQ ID NO.3; the terminator TrrnBT1 was selected, and its nucleotide sequence was SEQ ID NO.4; and the expression frame of the malonyl-CoA reductase gene MCR was obtained by PCR fusion, and its nucleotide sequence was SEQ ID NO.5.

选择启动子Pcpc560₆₈₀₃，其核苷酸序列SEQ ID NO.6；选择终止子TrbcL₆₈₀₃，其核苷酸序列SEQ ID NO.7；通过PCR融合获得丙二酸半醛还原酶基因MSR的表达框，其核苷酸序列SEQ ID NO.8。The promoter Pcpc560₆₈₀₃ was selected, and its nucleotide sequence was SEQ ID NO.6; the terminator TrbcL₆₈₀₃ was selected, and its nucleotide sequence was SEQ ID NO.7; and the expression frame of the malonate semialdehyde reductase gene MSR was obtained by PCR fusion, and its nucleotide sequence was SEQ ID NO.8.

通过Gibson组装方式将两个基因的表达框构建到质粒pRS416-kana上，并通过转入大肠杆菌DH5a感受态细胞中进行筛选，获得重组载体pRS416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃。空质粒和重组质粒的核苷酸序列分别是SEQ ID NO.9与SEQ ID NO.10。The expression frames of the two genes were constructed into the plasmid pRS416-kana by Gibson assembly, and the recombinant vector pRS416-kana-PcpcB1₇₉₄₂ -MCR-TrrnBT1-Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃ was obtained by transformation into E. coli DH5a competent cells for screening. The nucleotide sequences of the empty plasmid and the recombinant plasmid are SEQ ID NO.9 and SEQ ID NO.10, respectively.

2）将步骤1）获得的重组载体，转化到嗜热蓝细菌E542中，并通过卡那霉素的抗性筛选获得重组嗜热蓝细菌E542。由于PcpcB1₇₉₄₂与Pcpc560₆₈₀₃都是蓝细菌组成型启动子，因此MCR和MSR可以在发酵培养的过程中直接进行转录和翻译。2) The recombinant vector obtained in step 1) is transformed into thermophilic cyanobacteria E542, and recombinant thermophilic cyanobacteria E542 is obtained by kanamycin resistance screening. Since PcpcB1₇₉₄₂ and Pcpc560₆₈₀₃ are both cyanobacterial constitutive promoters, MCR and MSR can be directly transcribed and translated during the fermentation culture process.

本发明还提供了上述重组菌在发酵生产3-羟基丙酸中的应用。The present invention also provides the use of the recombinant bacteria in the fermentation production of 3-hydroxypropionic acid.

在本发明的一个实施例中，所述生产3-羟基丙酸的方法，步骤如下：In one embodiment of the present invention, the method for producing 3-hydroxypropionic acid comprises the following steps:

1）活化重组嗜热蓝细菌E542，获得种子菌液；1) Activate the recombinant thermophilic cyanobacterium E542 to obtain seed bacterial liquid;

2）将步骤1）所得的种子液接到含有卡纳霉素的BG11培养基中进行培养5-10天。2) The seed solution obtained in step 1) is inoculated into BG11 medium containing kanamycin and cultured for 5-10 days.

在本发明的一个实施例中，步骤2）的培养条件是55℃，220rpm，300μmol photons/m²/s。In one embodiment of the present invention, the culture conditions of step 2) are 55° C., 220 rpm, and 300 μmol photons/m² /s.

在本发明的一个实施例中，步骤2)中所述种子液按体积比为2%的接种量接种到含终浓度为30ug/mL卡纳霉素的BG11培养基中，并于55℃，220rpm，300μmol photons/m²/s的条件下振荡培养5-10天后终止发酵。In one embodiment of the present invention, the seed solution in step 2) is inoculated into BG11 medium containing kanamycin at a final concentration of 30 ug/mL at a volume ratio of 2%, and the fermentation is terminated after shaking culture at 55°C, 220 rpm, and 300 μmol photons/m² /s for 5-10 days.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1：pRSF416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃的重组载体谱；Figure 1: Recombinant vector spectrum of pRSF416-kana-PcpcB1₇₉₄₂ -MCR-TrrnBT1-Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃ ;

图2：重组嗜热蓝细菌E542直接利用废气二氧化碳生产3-羟基丙酸示意图；Figure 2: Schematic diagram of recombinant thermophilic cyanobacterium E542 directly utilizing waste gas carbon dioxide to produce 3-hydroxypropionic acid;

图3：重组嗜热蓝细菌E542发酵液中检测3-羟基丙酸的HPLC-Q-TOF-MS图。Figure 3: HPLC-Q-TOF-MS graph of detection of 3-hydroxypropionic acid in the fermentation broth of recombinant thermophilic cyanobacterium E542.

具体实施方式Detailed ways

下面通过实例来进一步阐明本发明，但本发明并不限于以下实施例。The present invention is further illustrated by examples below, but the present invention is not limited to the following embodiments.

在以下的实施例中，使用的实验方法若无特殊说明，均为常规方法。In the following examples, the experimental methods used are conventional methods unless otherwise specified.

在以下的实施例中，使用的材料、试剂等若无特殊说明，均可从商业途径获得。In the following examples, the materials and reagents used can be obtained from commercial sources unless otherwise specified.

所用的连接酶试剂购自中美泰和生物技术(北京)有限公司，质粒提取试剂盒和DNA纯化试剂盒购自南京诺唯赞生物科技股份有限公司。引物合成与质粒测序由北京擎科生物科技股份有限公司完成。所有培养基若无特殊说明均用去离子水配制。The ligase reagent used was purchased from Sino-US Taihe Biotechnology (Beijing) Co., Ltd., and the plasmid extraction kit and DNA purification kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd. Primer synthesis and plasmid sequencing were completed by Beijing Qingke Biotechnology Co., Ltd. All culture media were prepared with deionized water unless otherwise specified.

培养基配方：Culture medium formula:

1）培养基1) Culture medium

LB培养基：5g/L酵母提取物，10g/L氯化钠，10g/L蛋白胨，溶于一定体积的蒸馏水，于121℃条件下灭菌20min。LB medium: 5 g/L yeast extract, 10 g/L sodium chloride, and 10 g/L peptone were dissolved in a certain volume of distilled water and sterilized at 121°C for 20 min.

BG11培养基：1.7g/L BG11粉末溶于一定体积的蒸馏水，于121℃条件下灭菌20min。BG11 medium: 1.7 g/L BG11 powder was dissolved in a certain volume of distilled water and sterilized at 121°C for 20 min.

在实际培养过程中，需要向上述培养基中添加一定浓度的抗生素以维持游离质粒的稳定性，如30ug/mL卡纳霉素。In the actual culture process, a certain concentration of antibiotics needs to be added to the above culture medium to maintain the stability of the free plasmid, such as 30ug/mL kanamycin.

实施例1 重组载体pRS416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃构建Example 1 Construction of recombinant vector pRS416-kana-PcpcB1₇₉₄₂ -MCR-TrrnBT1-Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃

本实施例中，来源于嗜热金属球菌Metallosphaera sedula的丙二酰-CoA还原酶基因MCR（NCBI的蛋白质编号是A4YEN2.1）和丙二酸半醛还原酶基因MSR（NCBI的蛋白质编号是ABP96133.1）是通过全基因合成，MCR经过密码子优化后的核苷酸序列如SEQ ID NO.1所示；MSR经过密码子优化后的核苷酸序列如SEQ ID NO.2所示；启动和终止MCR表达的元件序列分别为SEQ ID NO.3和SEQ ID NO.4；启动和终止MSR表达的元件序列分别为SEQ ID NO.6和SEQ ID NO.7；载体为pRS416-kana，序列如SEQ ID NO.9所示。In this embodiment, the malonyl-CoA reductase gene MCR (the protein number of NCBI is A4YEN2.1) and the malonate semialdehyde reductase gene MSR (the protein number of NCBI is ABP96133.1) derived from the thermophilic metallosphaeraMetallosphaera sedula are synthesized by whole gene synthesis, and the nucleotide sequence of MCR after codon optimization is shown in SEQ ID NO.1; the nucleotide sequence of MSR after codon optimization is shown in SEQ ID NO.2; the element sequences for starting and stopping the expression of MCR are SEQ ID NO.3 and SEQ ID NO.4, respectively; the element sequences for starting and stopping the expression of MSR are SEQ ID NO.6 and SEQ ID NO.7, respectively; the vector is pRS416-kana, and the sequence is shown in SEQ ID NO.9.

将载体pRS416-kana通过KpnⅠ和XbaⅠ进行酶切获得线性化大片段；通过PCR融合分别组装丙二酰-CoA还原酶基因MCR表达框PcpcB1₇₉₄₂-MCR-TrrnBT1和丙二酸半醛还原酶基因MSR表达框Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃。将这三个片段通过酶连进行质粒构建。载体与插入片段（MCR、MSR）按照摩尔比1:2的比例，50℃连接15min，连接产物转化E. coliDH5a，然后涂布在50ug/mL卡纳霉素的LB固体平板上，利用PCR筛选阳性克隆。从阳性克隆提取重组质粒通过限制性酶切进行酶切验证和测序鉴定。重组质粒图谱如图1所示。The vector pRS416-kana was digested with KpnⅠ and XbaⅠ to obtain a linearized large fragment; the expression frame of the malonyl-CoA reductase gene MCR PcpcB1₇₉₄₂ -MCR-TrrnBT1 and the expression frame of the malonate semialdehyde reductase gene MSR Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃ were assembled by PCR fusion. The three fragments were linked to construct plasmids. The vector and the insert fragment (MCR, MSR) were connected at a molar ratio of 1:2 at 50℃ for 15min. The ligation product was transformed intoE. coli DH5a, and then coated on a LB solid plate containing 50ug/mL kanamycin, and positive clones were screened by PCR. The recombinant plasmid was extracted from the positive clone and digested by restriction enzymes for enzyme verification and sequencing. The recombinant plasmid map is shown in Figure 1.

实施例2 重组蓝细菌pRS416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃构建Example 2 Construction of recombinant cyanobacteria pRS416-kana-PcpcB1₇₉₄₂ -MCR-TrrnBT1-Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃

实施例1的重组质粒pRS416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃通过自然转化转入嗜热蓝细菌E542,然后涂布在30ug/mL卡纳霉素的BG11固体平板上，利用PCR筛选阳性克隆。从阳性克隆提取总gDNA进行测序鉴定。重组嗜热蓝细菌E542直接利用废气二氧化碳生产3-羟基丙酸示意图如图2所示。The recombinant plasmid pRS416-kana-PcpcB1₇₉₄₂ -MCR-TrrnBT1-Pcpc560₆₈₀₃ -MSR-TrbcL₆₈₀₃ of Example 1 was transferred into thermophilic cyanobacteria E542 by natural transformation, and then coated on a BG11 solid plate containing 30ug/mL kanamycin, and positive clones were screened by PCR. Total gDNA was extracted from the positive clones for sequencing and identification. The schematic diagram of recombinant thermophilic cyanobacteria E542 directly using waste gas carbon dioxide to produce 3-hydroxypropionic acid is shown in Figure 2.

实施例3 重组嗜热蓝细菌E542产3-羟基丙酸Example 3 Recombinant Thermophilic Cyanobacterium E542 Produces 3-Hydroxypropionic Acid

实施例2获得的单克隆重组嗜热蓝细菌E542在BG11中进行活化，获得种子液；将所得的种子液接到含有30ug/mL卡纳霉素的BG11培养基中，并于55℃，220rpm，300μmolphotons/m²/s条件下进行培养5-10天。The monoclonal recombinant thermophilic cyanobacterium E542 obtained in Example 2 was activated in BG11 to obtain seed solution; the obtained seed solution was inoculated into BG11 medium containing 30 ug/mL kanamycin and cultured at 55°C, 220 rpm, 300 μmolphotons/m² /s for 5-10 days.

取1mL发酵液，4℃，6000rpm离心10min，取上清，利用高效液相色谱-四极杆-飞行时间质谱 (HPLC-Q-TOF-MS) 联用检测产物。重组嗜热蓝细菌E542发酵液中检测3-羟基丙酸的HPLC-Q-TOF-MS图如图3所示。Take 1 mL of fermentation broth, centrifuge at 4°C, 6000 rpm for 10 min, take the supernatant, and detect the product using high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF-MS). The HPLC-Q-TOF-MS graph of 3-hydroxypropionic acid detected in the fermentation broth of recombinant thermophilic cyanobacterium E542 is shown in Figure 3.

发酵液中3-羟基丙酸的出峰时间是0.14分钟左右，负离子模式m/z为89.0，与3-羟基丙酸标准品一致。实验结果表明，重组嗜热蓝细菌E542可以直接利用废气二氧化碳产3-羟基丙酸。The peak time of 3-hydroxypropionic acid in the fermentation broth is about 0.14 minutes, and the m/z in negative ion mode is 89.0, which is consistent with the 3-hydroxypropionic acid standard. The experimental results show that the recombinant thermophilic cyanobacterium E542 can directly use waste gas carbon dioxide to produce 3-hydroxypropionic acid.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干优化改进和补充，这些改进和补充也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several optimization improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims (6)

1. A recombinant bacterium for producing 3-hydroxypropionic acid by directly utilizing waste carbon dioxide, which is characterized in that the recombinant bacterium is obtained by introducing a malonyl-CoA reductase encoding gene MCR and a malonate semialdehyde reductase encoding gene MSR into a cyanobacterium thermophilum E542 through a shuttle plasmid pRS 416-kana; the coding malonyl-CoA reductase gene MCR and the coding malonate semialdehyde reductase gene MSR are optimized in a codon preference manner based on thermophilic cyanobacteria E542, and the nucleotide sequence of the MCR after codon optimization is shown as SEQ ID NO. 1; the nucleotide sequence of the MSR after codon optimization is shown as SEQ ID NO. 2.

2. The recombinant bacterium of claim 1, wherein the malonyl-CoA reductase-encoding gene MCR and the malonate semialdehyde reductase-encoding gene MSR are both derived from metallococcus thermophilus Metallosphaera sedula.

3. The recombinant bacterium of claim 1, wherein the malonyl-CoA reductase has a protein number at NCBI of A4YEN2.1; the protein number of malonate semialdehyde reductase at NCBI is ABP96133.1.

4. A method of constructing a recombinant bacterium according to any one of claims 1 to 3, comprising the steps of:

1) Synthesizing a malonyl-CoA reductase gene MCR and a malonate semialdehyde reductase gene MSR respectively;

selecting a promoter PcpcB to₇₉₄₂, the nucleotide sequence of which is SEQ ID NO.3; selecting terminator TrrnBT, its nucleotide sequence SEQ ID NO.4; obtaining an expression frame of malonyl-CoA reductase gene MCR through PCR fusion, wherein the nucleotide sequence of the expression frame is SEQ ID NO.5;

Selecting a promoter Pcpc560,560, 560₆₈₀₃, the nucleotide sequence of which is SEQ ID NO.6; selecting terminator TrbcL₆₈₀₃ whose nucleotide sequence is SEQ ID NO.7; fusion of the expression cassette of malonate semialdehyde reductase gene MSR by PCR, the nucleotide sequence SEQ ID NO.8;

Constructing expression frames of two genes on plasmid pRS416-kana by a Gibson assembly mode, and carrying out screening by transferring into competent cells of escherichia coli DH5a to obtain recombinant vector pRS416-kana-PcpcB1₇₉₄₂-MCR-TrrnBT1-Pcpc560₆₈₀₃-MSR-TrbcL₆₈₀₃; the nucleotide sequences of the empty plasmid and the recombinant plasmid are SEQ ID NO.9 and SEQ ID NO.10 respectively;

2) Transforming the recombinant vector obtained in the step 1) into a thermophilic cyanobacteria E542, and obtaining the recombinant thermophilic cyanobacteria E542 through resistance screening of kanamycin; since PcpcB,₇₉₄₂ and Pcpc, 560 and₆₈₀₃ are cyanobacteria constitutive promoters, MCR and MSR are transcribed and translated directly during fermentation culture.

5. Use of a recombinant bacterium according to any one of claims 1-3 in a fermentation process for producing 3-hydroxypropionic acid.

6. The use according to claim 5, characterized in that the process for producing 3-hydroxypropionic acid comprises the following steps:

1) Activating recombinant thermophilic cyanobacteria E542 to obtain seed bacterial liquid;

2) The seed bacterial liquid obtained in the step 1) is inoculated into a BG11 medium containing 30ug/mL of the calicheamicin, and cultured for 5-10 days under the conditions of 55 ℃ and 220rpm and 300 mu mol photons/m²/s.