技术领域technical field
本发明涉及一株含特定甘油脱氢酶基因序列的氧化葡萄糖酸杆菌，以及催化甘油转化为1，3-二羟基丙酮的应用，属于生物技术领域。The invention relates to a gluconobacterium oxydans containing a specific glycerol dehydrogenase gene sequence and the application of catalyzing the conversion of glycerol into 1,3-dihydroxyacetone, which belongs to the field of biotechnology.
背景技术Background technique
1，3-二羟基丙酮(1，3-Dihydroxyacetone)，简称DHA，简单分子式为C3H6O6，是结构最简单的多羟基酮糖，相对分子质量是90.08。纯品呈白色或类白色粉末状晶体，带甜味和特殊气味，易溶于水、乙醇、丙酮和乙醚等有机溶剂，化学性质活泼，在化工、医药、食品、化妆品等领域应用广泛。1,3-Dihydroxyacetone (1,3-Dihydroxyacetone), referred to as DHA, has a simple molecular formula of C3 H6 O6 , and is the simplest polyhydroxy ketose sugar, with a relative molecular mass of 90.08. The pure product is white or off-white powder crystal, with sweet taste and special smell, easily soluble in water, ethanol, acetone, ether and other organic solvents, with active chemical properties, widely used in chemical industry, medicine, food, cosmetics and other fields.
目前DHA的制备主要有化学合成法和微生物酶催化法两类。化学催化法需要多步化学反应、金属催化剂价格昂贵，原料利用率低，副产物多，分离纯化困难，环境污染严重。而微生物酶催化法则具有反应立体选择性高、反应条件温和、原料利用率高、工艺简单、环境友好等特点，因此更受青睐。At present, the preparation of DHA mainly includes chemical synthesis and microbial enzyme catalysis. Chemical catalysis requires multi-step chemical reactions, expensive metal catalysts, low raw material utilization, many by-products, difficult separation and purification, and serious environmental pollution. The microbial enzyme catalysis method has the characteristics of high reaction stereoselectivity, mild reaction conditions, high utilization rate of raw materials, simple process, and environmental friendliness, so it is more popular.
氧化葡萄糖酸杆菌是专性好氧的微生物，其细胞膜表面的一些特殊的氧化还原酶能够不完全氧化一些糖、醇和酸。在工业上利用膜结合甘油脱氢酶催化甘油产生DHA的特性，氧化葡萄糖酸杆菌成为产生DHA的主要菌种，但是，底物甘油和产物DHA均会对菌体的生长和DHA的转化产生抑制作用。为了解决这些问题，研究人员通过改变DHA的转化方式、改进发酵设备和控制溶氧、pH条件等方法，包括利用静息细胞、流加发酵工艺、固定化细胞和多阶反应器等降低高浓度底物和产物对菌体生长和DHA转化的抑制作用。如Hekmat等人(Process Biochemistry，2007，42：71-76)通过固定化细胞和半连续重复补料的发酵方法减少底物和产物抑制，在填充床鼓泡反应器发酵18天后，固定化细胞产生的生物量可达发酵罐内总生物量的65％；同时，使用优化培养基后产量提高了76％；对比于滴流床反应器，空时产量提高了3.7倍。ka等人(Collection of Czechoslovak ChemicalCommunications，2007，72：1269-1283)在恒温恒容分批反应器，凝胶包埋固定化氧化葡萄糖酸杆菌细胞转化甘油生成DHA。随反应中DHA浓度的提高，产物抑制作用不可避免。Wei等人(Preparative Biochemistry&Biotechnology，2007，37(1)：61-67)用聚乙烯醇固定化氧化葡萄糖酸杆菌细胞，于30℃和pH 6.0条件下发酵，发现在第14天时菌体仍保持高活性，且仅有10％的活性损失，DHA的平均转化率达到86％；1.5L的搅拌反应器中，平均转化率约为86％。Hu等人(Biotechnology and Bioprocess Engineering，2010，15：651-656)在溶氧参数驱动的补料分批发酵罐中，通过调控pH来提高DHA的产量。在30％空气饱和度下，前20个小时维持pH 6.0，后调节至5.0时，维持80％甘油浓度补料，在72h时得到最优DHA浓度是175.9±6.7g/L。Gluconobacter oxidans is an obligate aerobic microorganism, and some special oxidoreductases on the surface of its cell membrane can incompletely oxidize some sugars, alcohols and acids. In the industry, the membrane-bound glycerol dehydrogenase catalyzes the characteristics of glycerol to produce DHA. Gluconobacter oxidans has become the main strain for producing DHA. However, both the substrate glycerol and the product DHA will inhibit the growth of the bacteria and the transformation of DHA. effect. In order to solve these problems, researchers have changed the conversion mode of DHA, improved fermentation equipment, and controlled dissolved oxygen and pH conditions, including the use of resting cells, fed-batch fermentation processes, immobilized cells, and multi-stage reactors to reduce high concentrations. Inhibitory effects of substrates and products on cell growth and DHA transformation. For example, Hekmat et al. (Process Biochemistry, 2007, 42:71-76) reduced substrate and product inhibition by immobilized cells and semi-continuous repeated feeding fermentation methods. After 18 days of fermentation in a packed bed bubble reactor, immobilized cells The biomass produced can reach 65% of the total biomass in the fermenter; at the same time, the yield is increased by 76% after using the optimized medium; compared with the trickle bed reactor, the space-time yield is increased by 3.7 times. Ka et al. (Collection of Czechoslovak Chemical Communications, 2007, 72: 1269-1283) converted glycerol to DHA with gel-embedded and immobilized Gluconobacter oxidans cells in a constant temperature and constant volume batch reactor. With the increase of DHA concentration in the reaction, product inhibition is inevitable. Wei et al. (Preparative Biochemistry&Biotechnology, 2007, 37(1): 61-67) immobilized Gluconobacter oxidans cells with polyvinyl alcohol and fermented them at 30°C and pH 6.0, and found that the cells remained high on the 14th day. Activity, and only 10% activity loss, the average conversion rate of DHA reaches 86%; In the stirred reactor of 1.5L, the average conversion rate is about 86%. Hu et al. (Biotechnology and Bioprocess Engineering, 2010, 15:651-656) increased the production of DHA by adjusting pH in a fed-batch fermenter driven by dissolved oxygen parameters. Under 30% air saturation, the pH was maintained at 6.0 for the first 20 hours, and when it was adjusted to 5.0, 80% glycerol was fed. The optimal DHA concentration was 175.9±6.7g/L at 72 hours.
氧化葡萄糖酸杆菌的膜结合甘油脱氢酶催化甘油生成DHA，依赖于吡咯喹啉醌(Pyrroloquinoline quinine，PQQ)作为辅酶。Tatsuo Hoshino等人(Biochimica etBiophysica Acta，2003，1647：278-288)尝试从G.suboxydans IFO 3255中纯化并研究了膜结合山梨醇脱氢酶的酶学性质，对其基因进行克隆表达后发现，G.suboxydans IFO 3255中分离的膜结合山梨醇脱氢酶和G.Industrius IFO 3266中纯化的膜结合甘油脱氢酶基本一致，也能催化甘油生成DHA。膜结合山梨醇脱氢酶由两个亚基(SldAB)构成，sldA作为结构基因，由2220对碱基构成，编码740个氨基酸残基。sldB是sldA基因上游的一段开放阅读框，由381对碱基构成，编码126个氨基酸残基，分子量为13735Da。正如对Gluconobacter oxydans621H全基因组测序中可知(Nature Biotechnology，2005，23(2)：195-200)，G.oxydans细胞膜上的膜结合甘油/山梨醇脱氢酶(sldAB，GOX 854-855)能催化D-山梨醇、葡萄糖酸和甘油生成相应的L-山梨糖、5-酮葡萄糖酸和DHA。由6enBank中查询的序列可知，sldA由2232对碱基构成，编码743个氨基酸残基的甘油脱氢酶的大亚基。sldB在sldA上游，由381对碱基构成，编码126个氨基酸残基。Membrane-bound glycerol dehydrogenase of Gluconobacter oxidans catalyzes the production of DHA from glycerol, which relies on pyrroloquinoline quinine (PQQ) as a coenzyme. Tatsuo Hoshino et al. (Biochimica et Biophysica Acta, 2003, 1647: 278-288) attempted to purify and study the enzymatic properties of membrane-bound sorbitol dehydrogenase from G.suboxydans IFO 3255. After cloning and expressing its gene, it was found that The membrane-bound sorbitol dehydrogenase isolated from G.suboxydans IFO 3255 is basically the same as the membrane-bound glycerol dehydrogenase purified from G.Industrius IFO 3266, which can also catalyze the production of DHA from glycerol. Membrane-bound sorbitol dehydrogenase consists of two subunits (SldAB), and sldA, as a structural gene, consists of 2220 base pairs and encodes 740 amino acid residues. sldB is an open reading frame upstream of the sldA gene, which consists of 381 base pairs, encodes 126 amino acid residues, and has a molecular weight of 13735Da. As known from the whole genome sequencing of Gluconobacter oxydans621H (Nature Biotechnology, 2005, 23(2):195-200), membrane-bound glycerol/sorbitol dehydrogenase (sldAB, GOX 854-855) on the cell membrane of G.oxydans can catalyze D-sorbitol, gluconic acid and glycerol yield the corresponding L-sorbose, 5-ketogluconic acid and DHA. According to the queried sequence in 6enBank, sldA consists of 2232 base pairs and encodes the large subunit of glycerol dehydrogenase with 743 amino acid residues. sldB is upstream of sldA, consisting of 381 base pairs, encoding 126 amino acid residues.
近年来，通过生物诱变菌株的手段，能有效提高产物DHA的产量，使膜结合甘油脱氢酶发生突变，提高催化效率。如文献中报道，Hu和Zheng等人(Applied Biochemistry&Biotechnology，2011，165：1152-1160)利用紫外诱变G.oxydans ZJB11001，获得了摇瓶培养64h，DHA产量达40g/L的突变株；Hu(Preparative biochemistry&biotechnology，2012，42：15-28)的另一项离子束注入研究所得到的G.oxydans ZJB09113，经24h转化，DHA的产量为13.8g/L，比对照菌提高了2倍；马丽娟等(Biochemical engineering journal，2010，49：61-67)通过He-Ne激光辐射来诱变菌株，得到对甘油和DHA有抗性的正突变株G.oxydans6M51，培养30h的突变株的甘油脱氢酶比亲本菌株提高了75.17％活性，GM51的培养发酵时间缩短了16h，而DHA的产量提高到91.5％，在7L的发酵罐中培养DHA产量提高了77.6％。也有文献报道，应用基因重组技术，建立提高产物DHA的产量的途径。如(AppliedMicrobiology and Biotechnology，2007，76：553-559)考察了重组甘油脱氢酶基因工程菌在甘油培养基中生长和催化能力，不但提高催化活性，也增加了生物量。通过高效表达sldAB基因提高了DHA的产量，550mM甘油底物浓度下，野生菌株的DHA产率为36％-51％，而突变菌株为64％。方柏山等人(中国专利CN101418274[P])构建了含有克雷伯氏肺炎球菌DSM 1115的gldA基因重组载体pET-32gldA，将其转化至E.coli BL21后，获得了高表达甘油脱氢酶活性的重组大肠杆菌甘油脱氢酶基因工程菌，利用乳糖诱导OD600在0.4到1.2之间的重组菌高效表达重组甘油脱氢酶。在大肠杆菌中共表达甘油脱氢酶基因和其他酶的基因，也能提高微生物对甘油的利用。如魏淼等人(生物加工过程，2011，9(5)：59-64)通过在大肠杆菌中共表达gldA基因和二羟丙酮激酶基因(dhaKLM)提高了大肠杆菌在好氧条件下的利用甘油合成菌体的效率和厌氧条件下代谢甘油能力。故利用基因工程的方法，能有效提高甘油脱氢酶的表达以及微生物转化甘油生成DHA的效率。李明华等人(BioresourceTechnology，2010，101(21)：8294-8299)在膜结合甘油脱氢酶缺失的G.oxydans M5AM菌株中过表达甘油脱氢酶后，在pH调控且连续供氧的5L发酵罐条件下，100g/L甘油浓度下，和对照野生菌株相比，发酵8h后，DHA产量由约50％提高至96％，生物量由1.0g/gCDW/h提高至2.4g/gCDW/h，以静息细胞状态在180g/L甘油下，18h内积累了153g/L DHA。In recent years, by means of biological mutagenesis strains, the yield of the product DHA can be effectively increased, the membrane-bound glycerol dehydrogenase can be mutated, and the catalytic efficiency can be improved. As reported in the literature, Hu and Zheng et al. (Applied Biochemistry & Biotechnology, 2011, 165: 1152-1160) used ultraviolet mutagenesis to G. oxydans ZJB11001, and obtained a mutant strain whose DHA production reached 40 g/L in shake flask culture for 64 hours; Hu( G.oxydans ZJB09113 obtained from another ion beam implantation study of Preparative biochemistry&biotechnology, 2012, 42:15-28, after 24 hours of transformation, the production of DHA was 13.8g/L, which was 2 times higher than that of the control bacteria; Ma Lijuan et al. (Biochemical engineering journal, 2010, 49:61-67) He-Ne laser radiation was used to mutate the strain to obtain a positive mutant strain G.oxydans6M51 resistant to glycerol and DHA, and the glycerol dehydrogenase of the mutant strain cultivated for 30 hours Compared with the parent strain, the activity is increased by 75.17%, the culture and fermentation time of GM51 is shortened by 16h, and the output of DHA is increased to 91.5%, and the output of DHA in a 7L fermenter is increased by 77.6%. There are also reports in the literature that the application of gene recombination technology to establish a way to improve the yield of the product DHA. like (Applied Microbiology and Biotechnology, 2007, 76:553-559) investigated the growth and catalytic ability of recombinant glycerol dehydrogenase genetically engineered bacteria in glycerol medium, which not only improved catalytic activity, but also increased biomass. The production of DHA is improved by highly expressing the sldAB gene. Under the substrate concentration of 550mM glycerol, the DHA production rate of the wild strain is 36%-51%, while that of the mutant strain is 64%. Fang Baishan et al. (Chinese patent CN101418274[P]) constructed the gldA gene recombinant vector pET-32gldA containing Klebsiella pneumoniae DSM 1115, and transformed it into E.coli BL21 to obtain a highly expressed glycerol dehydrogenase Active recombinant Escherichia coli glycerol dehydrogenase genetically engineered bacteria, using lactose to induce recombinant bacteria with anOD600 between 0.4 and 1.2 to highly express recombinant glycerol dehydrogenase. Co-expression of the glycerol dehydrogenase gene and genes for other enzymes in E. coli also improved the utilization of glycerol by the microorganism. For example, Wei Miao et al. (Bioprocessing, 2011, 9(5): 59-64) improved the glycerol utilization of E. coli under aerobic conditions by co-expressing the gldA gene and the dihydroxyacetone kinase gene (dhaKLM) in E. coli Efficiency of synthetic bacteria and ability to metabolize glycerol under anaerobic conditions. Therefore, the use of genetic engineering methods can effectively improve the expression of glycerol dehydrogenase and the efficiency of microbial conversion of glycerol to DHA. Li Minghua et al. (BioresourceTechnology, 2010, 101(21): 8294-8299) overexpressed glycerol dehydrogenase in the membrane-bound glycerol dehydrogenase-deficient G.oxydans M5AM strain, and then fermented it in a 5L fermentation with pH regulation and continuous oxygen supply. Under tank conditions, at 100g/L glycerol concentration, compared with the control wild strain, after 8 hours of fermentation, the DHA production increased from about 50% to 96%, and the biomass increased from 1.0g/gCDW/h to 2.4g/gCDW/h , 153g/L DHA was accumulated within 18h under 180g/L glycerol in resting cell state.
从氧化葡萄糖酸杆菌膜组分中纯化膜结合甘油脱氢酶，分离纯化过程繁琐、酶活力损失大，同时，只有在添加了PQQ后，膜结合甘油脱氢酶才有全酶活性。膜结合甘油脱氢酶的三维结构以及三维结构与功能的关系规律没有被阐明。Purification of membrane-bound glycerol dehydrogenase from the membrane fraction of Gluconobacter oxydans is cumbersome, and the enzyme activity is greatly lost. At the same time, only after adding PQQ, the membrane-bound glycerol dehydrogenase has full enzyme activity. The three-dimensional structure of membrane-bound glycerol dehydrogenase and the relationship between three-dimensional structure and function have not been elucidated.
综上所述，通过改变DHA的生产方式、改进发酵设备和控制溶氧、pH条件等方法，包括利用静息细胞、流加发酵工艺、固定化细胞和多阶反应器等，以及膜结合甘油脱氢酶的基因重组操作，能有效降低了底物甘油和产物DHA对菌体的生长和DHA的转化的抑制作用。但是，目前缺乏膜结合甘油脱氢酶的三维结构以及三维结构与功能的关系规律研究基础，因而无法进行理性的结构改造和酶学性质提升；即便如此，仅仅开展膜结合甘油脱氢酶的研究对于进一步降低了底物甘油和产物DHA对菌体的生长和DHA的转化产生的抑制作用还是不够的。本发明通过菌种选育和基因操作，获得了一株含有特定核苷酸序列甘油脱氢酶基因、能够耐高底物甘油和产物的底盘细胞氧化葡萄糖酸杆菌I-2-239，并具有高效催化甘油产生DHA的应用价值。In summary, by changing the production method of DHA, improving fermentation equipment and controlling dissolved oxygen and pH conditions, including the use of resting cells, fed-batch fermentation processes, immobilized cells and multi-stage reactors, and membrane-bound glycerol The gene recombination operation of the dehydrogenase can effectively reduce the inhibitory effect of the substrate glycerol and the product DHA on the growth of bacteria and the transformation of DHA. However, there is currently a lack of research basis for the three-dimensional structure of membrane-bound glycerol dehydrogenase and the relationship between three-dimensional structure and function, so it is impossible to carry out rational structural modification and enzymatic property improvement; even so, only research on membrane-bound glycerol dehydrogenase It is not enough to further reduce the inhibitory effect of substrate glycerol and product DHA on the growth of thalline and the transformation of DHA. The present invention obtains a chassis cell Gluconobacter oxidans I-2-239 which contains a specific nucleotide sequence glycerol dehydrogenase gene and can tolerate high substrate glycerol and products through strain selection and genetic manipulation, and has The application value of efficiently catalyzing glycerol to produce DHA.
发明内容Contents of the invention
本发明的目的是提供一株含特定膜结合甘油脱氢酶基因序列的氧化葡萄糖酸杆菌，具有耐受高浓度底物甘油和产物DHA对菌体的生长以及DHA的转化产生抑制作用的特点，并具有高效催化甘油产生DHA的应用价值。The object of the present invention is to provide a strain of Gluconobacter oxydans containing a specific membrane-bound glycerol dehydrogenase gene sequence, which has the characteristics of being able to tolerate high concentrations of substrate glycerol and product DHA to inhibit the growth of the thallus and the transformation of DHA. And it has the application value of efficiently catalyzing glycerin to produce DHA.
本发明对一株从土壤中筛选菌株L-6，在30℃，200rpm条件下，发酵培养54h，DHA产量最高达47.98g/L。对其16S rDNA进行测序后，进行系谱树分析(图1)，结合菌落特征、理化分析，可将该菌株确定为氧化葡萄糖酸杆菌。The present invention selects a strain L-6 from the soil, ferments and cultures it for 54 hours under the conditions of 30° C. and 200 rpm, and the DHA output is up to 47.98 g/L. After its 16S rDNA was sequenced, the pedigree analysis was carried out (Fig. 1). Combined with the colony characteristics and physical and chemical analysis, the strain could be identified as Gluconobacter oxidans.
SEQ ID No.1所示为16S rDNA序列，为该菌株的定种提供了分子生物学依据。The 16S rDNA sequence shown in SEQ ID No.1 provides a molecular biological basis for the identification of the strain.
上述筛选菌株L-6经过多轮紫外诱变，得到遗传稳定的突变株U-2-115，经摇瓶发酵培养，在80g/L底物甘油下，DHA平均产量达71.40g/L，与L-6比较，产量提高48.8％。The above screened strain L-6 underwent multiple rounds of ultraviolet mutagenesis to obtain a genetically stable mutant strain U-2-115, which was fermented and cultured in shake flasks. Under the substrate glycerol of 80g/L, the average output of DHA reached 71.40g/L, which was comparable to that of Compared with L-6, the yield increased by 48.8%.
突变株U-2-115再经过室温等离子诱变，得到新的突变株A-2-64，在100g/L甘油浓度条件下，摇瓶发酵培养的DHA平均产量上升到90.2g/L，与突变株U-2-115和L-6比较，DHA产量分别提高26.3％和87.9％。Mutant strain U-2-115 undergoes plasma mutagenesis at room temperature to obtain a new mutant strain A-2-64. Under the condition of 100g/L glycerol concentration, the average yield of DHA in shake flask fermentation culture rises to 90.2g/L, which is the same as Compared with mutant strains U-2-115 and L-6, the DHA production was increased by 26.3% and 87.9%, respectively.
经过离子注入诱变和高通量筛选，在120g/L甘油底物浓度下，突变株I-2-239摇瓶发酵培养的DHA平均产量达103.5g/L，与突变株A-2-64和L-6比较，DHA产量分别提高14.7％和115.7％。After ion implantation mutagenesis and high-throughput screening, at a substrate concentration of 120g/L glycerol, the average DHA yield of the mutant strain I-2-239 shake flask fermentation culture reached 103.5g/L, which was comparable to that of the mutant strain A-2-64. Compared with L-6, the production of DHA increased by 14.7% and 115.7%, respectively.
与L-6比较，尽管突变株I-2-239在发酵结束时的生物量没有显著增加，但是突变株I-2-239在发酵培养基的生长速率明显加快，说明该突变株耐高甘油浓度的能力增强，最终导致发酵周期缩短。与此同时，突变株I-2-239的甘油脱氢酶活性显著增强。Compared with L-6, although the biomass of the mutant strain I-2-239 did not increase significantly at the end of the fermentation, the growth rate of the mutant strain I-2-239 in the fermentation medium was significantly accelerated, indicating that the mutant strain is resistant to high glycerol The ability to increase the concentration will eventually lead to a shortened fermentation period. At the same time, the glycerol dehydrogenase activity of the mutant strain I-2-239 was significantly enhanced.
上述高产DHA的氧化葡萄糖酸杆菌突变株的膜结合甘油脱氢酶特征如下(1)(2)(3)(4)所示：The membrane-bound glycerol dehydrogenase characteristics of the above-mentioned high-yield DHA-producing Gluconobacter oxidans mutant strain are as follows (1) (2) (3) (4):
(1)SEQ ID No.2是氧化葡萄糖酸杆菌突变株膜结合甘油脱氢酶特定的编码基因序列；(1) SEQ ID No.2 is the specific coding gene sequence of the membrane-bound glycerol dehydrogenase of the mutant strain of Gluconobacter oxidans;
(2)SEQ ID No.3是氧化葡萄糖杆菌突变株膜结合甘油脱氢酶小亚基SldB的特定的氨基酸序列；(2) SEQ ID No.3 is the specific amino acid sequence of the membrane-bound glycerol dehydrogenase small subunit SldB of the mutant strain of Gluconobacter oxidans;
(3)SEQ ID No.4是氧化葡萄糖酸杆菌突变株膜结合甘油脱氢酶大亚基SldA特定的氨基酸序列；(3) SEQ ID No.4 is the specific amino acid sequence of the membrane-bound glycerol dehydrogenase large subunit SldA of the mutant strain of Gluconobacter oxidans;
(4)SEQ ID No.2的特征在于膜结合甘油脱氢酶特定的编码基因序列由2232bp的sldA和381bp的sldB两部分组成，sldB的终止密码子和sldA的起始密码子重叠1个碱基，其中一个或几个核苷酸的替代引起相应的氨基酸残基的取代，影响其膜结合甘油脱氢酶的功能。(4) SEQ ID No.2 is characterized in that the specific coding gene sequence of membrane-bound glycerol dehydrogenase is composed of 2232bp sldA and 381bp sldB, and the stop codon of sldB overlaps with the start codon of sldA by 1 base base, wherein the substitution of one or several nucleotides causes the substitution of the corresponding amino acid residues, affecting its membrane-bound glycerol dehydrogenase function.
(5)SEQ ID No.4所示氨基酸序列中，SldA是催化亚基，编码743个氨基酸，SEQ ID No.3所示的序列中，SldB可能是帮助催化亚基折叠成熟的小亚基，有126个残基。(5) In the amino acid sequence shown in SEQ ID No.4, SldA is the catalytic subunit, encoding 743 amino acids. In the sequence shown in SEQ ID No.3, SldB may be a small subunit that helps the catalytic subunit fold and mature, There are 126 residues.
上述酶的编码基因序列和氨基酸序列属于本发明保护范围。The coding gene sequence and amino acid sequence of the above enzymes belong to the protection scope of the present invention.
上述突变菌株的甘油脱氢酶基因的特定核苷酸序列经过测序发现，野生菌株L-6和突变株I-2-239的甘油脱氢酶基因存在11个核苷酸差异(图2)，其中10个核苷酸差异导致了编码氨基酸的密码子改变，其中3个在sldB上，7个在sldA上(表1)。The specific nucleotide sequence of the glycerol dehydrogenase gene of the above-mentioned mutant strains was sequenced and found that there were 11 nucleotide differences in the glycerol dehydrogenase gene of the wild strain L-6 and the mutant strain I-2-239 (Fig. 2), Ten of these nucleotide differences resulted in codon changes encoding amino acids, 3 of which were in sldB and 7 in sldA (Table 1).
表1突变菌株和出发菌株之间甘油脱氢酶的突变位点对比Table 1 Comparison of mutation sites of glycerol dehydrogenase between mutant strains and starting strains
对突变菌株膜结合甘油脱氢酶进行同源建模，得到图3所示的三维结构，进行结构与功能关系分析发现，突变株在甘油脱氢酶辅酶PQQ结合位点上或附近有5个氨基酸残基(分别是第270，289，364，367和434位)突变。Homology modeling was carried out on the membrane-bound glycerol dehydrogenase of the mutant strain, and the three-dimensional structure shown in Figure 3 was obtained. The analysis of the relationship between structure and function found that the mutant strain had 5 binding sites on or near the coenzyme PQQ binding site of glycerol dehydrogenase. Amino acid residues (positions 270, 289, 364, 367 and 434, respectively) were mutated.
上述的这些残基的突变位点可能导致甘油脱氢酶与PQQ的结合程度发生变化，加快氧化还原的电子传递效率，进而提高了催化效率。The above-mentioned mutation sites of these residues may lead to changes in the binding degree of glycerol dehydrogenase and PQQ, accelerate the redox electron transfer efficiency, and then improve the catalytic efficiency.
以上述氧化葡萄糖酸杆菌突变株的甘油脱氢酶两个亚基基因的上下游序列为引物，进行实时荧光定量PCR进行转录水平的分析发现，尽管突变株的生物量没有显著增加，但是，甘油脱氢酶的转录水平提高，影响了甘油脱氢酶的表达量，进而使单位菌体的催化活性得到显著提高。Using the upstream and downstream sequences of the two subunit genes of glycerol dehydrogenase of the above-mentioned Gluconobacter oxidans mutant strain as primers, real-time fluorescent quantitative PCR was carried out to analyze the transcription level, and it was found that although the biomass of the mutant strain did not increase significantly, glycerol The increase of the transcription level of dehydrogenase affects the expression of glycerol dehydrogenase, and then the catalytic activity of the unit cell is significantly improved.
利用基因工程手段可以将该高产DHA的膜结合甘油脱氢酶编码基因序列插入载体后形成重组质粒，再导入相应的宿主细胞构建重组菌。By means of genetic engineering, the high-yield DHA membrane-bound glycerol dehydrogenase coding gene sequence can be inserted into a vector to form a recombinant plasmid, and then introduced into a corresponding host cell to construct a recombinant bacterium.
(1)在大肠杆菌中表达插入该高产菌株的膜结合甘油脱氢酶和PQQ合成酶的编码基因的重组质粒，诱导表达后能够转化甘油产生DHA。(1) Express in E. coli a recombinant plasmid inserted into the coding genes of the membrane-bound glycerol dehydrogenase and PQQ synthase of the high-yielding strain, which can convert glycerol to produce DHA after induced expression.
(2)根据高产DHA的突变菌株膜结合甘油脱氢酶编码基因设计引物，PCR扩增该基因，酶切后插入载体pBBR1MCS-2，构建的重组质粒转化E.coli DH5α，扩增细胞，提取重组质粒，转化氧化葡萄糖酸杆菌，构建的重组菌高效转化甘油生成DHA。(2) Design primers according to the membrane-bound glycerol dehydrogenase coding gene of the mutant strain with high production of DHA, amplify the gene by PCR, insert the vector pBBR1MCS-2 after digestion, transform the recombinant plasmid into E.coli DH5α, amplify the cells, and extract Recombinant plasmids are transformed into Gluconobacter oxidans, and the constructed recombinant bacteria efficiently transform glycerol to produce DHA.
本发明的有益效果：Beneficial effects of the present invention:
本发明能提供一株含特定膜结合甘油脱氢酶基因序列的氧化葡萄糖酸杆菌，主要表现为：(1)在发酵培养基中的生长速率明显加快，说明该突变株耐高甘油浓度的能力增强，最终导致发酵周期缩短；(2)甘油脱氢酶基因转录水平、甘油脱氢酶酶活性显著提高；(3)甘油脱氢酶基因序列的突变可能导致了辅酶PQQ和酶结合程度的改变，加快氧化还原的电子传递效率。因此，本发明能够提供一株高效催化甘油，生成DHA的氧化葡萄糖酸杆菌；利用基因工程手段，可以构建该高产DHA的膜结合甘油脱氢酶的重组质粒，进而导入大肠杆菌或氧化葡萄糖酸杆菌中进行表达，进一步提高转化甘油生成DHA的效能。The present invention can provide a strain of Gluconobacter oxydans containing a specific membrane-bound glycerol dehydrogenase gene sequence, which is mainly characterized by: (1) the growth rate in the fermentation medium is obviously accelerated, indicating the ability of the mutant strain to withstand high glycerol concentration (2) Glycerol dehydrogenase gene transcription level and glycerol dehydrogenase enzyme activity were significantly increased; (3) The mutation of glycerol dehydrogenase gene sequence may lead to the change of coenzyme PQQ and enzyme binding degree , to accelerate the redox electron transfer efficiency. Therefore, the present invention can provide a high-efficiency catalyzed glycerol to produce DHA Gluconobacter oxidans; using genetic engineering means, the recombinant plasmid of the high-yield DHA membrane-bound glycerol dehydrogenase can be constructed, and then introduced into Escherichia coli or Gluconobacter oxidans Expressed in the medium to further improve the efficiency of converting glycerol into DHA.
附图说明Description of drawings
图1：菌株L-6和相关菌株的系统发育树Figure 1: Phylogenetic tree of strain L-6 and related strains
图2：突变菌株I-2-239和出发菌株L-6的膜结合甘油脱氢酶基因序列(sldAB)比较。黑框表示两菌株的基因序列的碱基差异。实心箭头表示sldB的起始端，两条粗线之间是sldB的编码区域；空心箭头表示sldA的起始端，两条细线之间是sldA的编码区域。Figure 2: Comparison of the membrane-bound glycerol dehydrogenase gene sequence (sldAB) of the mutant strain I-2-239 and the starting strain L-6. The black boxes indicate the base differences in the gene sequences of the two strains. The solid arrow indicates the beginning of sldB, and the coding region of sldB is between the two thick lines; the hollow arrow indicates the beginning of sldA, and the coding region of sldA is between the two thin lines.
图3：由I-TASSER生成的突变菌株I-2-239的甘油脱氢酶的3D结构图。黑色螺旋指的是α螺旋结构，浅灰色飘带模型指的是β片层结构，β转角以深灰色细线显示，无规卷曲结构以浅灰色细线模型显示。Figure 3: 3D structure diagram of glycerol dehydrogenase of mutant strain I-2-239 generated by I-TASSER. The black helix refers to the α-helix structure, the light gray ribbon model refers to the β-sheet structure, the β-turn is shown as a dark gray thin line, and the random coil structure is shown as a light gray thin line model.
具体实施方式Detailed ways
根据下述实施案例，对本菌株用于高浓度甘油下产DHA的应用及其膜结合甘油脱氢酶的基因序列的作用会有更好的理解。According to the following examples, the application of this strain to produce DHA under high concentration of glycerol and the role of the gene sequence of membrane-bound glycerol dehydrogenase will be better understood.
下述实施例中的实验方法，如无特殊说明，均为常规方法。材料和试剂等，如无特殊说明，除菌体为本实验室保藏外，其余均为商业途径得到。下述实施例中涉及到的发酵、检测和构建重组载体，如无特殊说明，是结合前人发明研究和本菌株的实际应用过程，仅用于说明本菌株的应用，而不应当也不会限制权利要求书中所详细描述的本发明。The experimental methods in the following examples are conventional methods unless otherwise specified. Materials and reagents, etc., unless otherwise specified, were obtained from commercial channels except for the bacteria stored in our laboratory. The fermentation, detection and construction of recombinant vectors involved in the following examples, unless otherwise specified, are combined with previous invention research and the actual application process of this strain, and are only used to illustrate the application of this strain, and should not and will not The invention described in detail in the claims is limited.
实施例1：野生株和突变株的发酵转化甘油生成DHA的比较Embodiment 1: the comparison of the fermentative conversion glycerol of wild strain and mutant strain to produce DHA
种子培养基(g/L)：甘油20，葡萄糖20，酵母膏5Seed medium (g/L): glycerol 20, glucose 20, yeast extract 5
固体平板培养基(g/L)：甘油20，葡萄糖20，酵母粉5，CaCO35，琼脂粉20Solid plate medium (g/L): glycerol 20, glucose 20, yeast powder 5, CaCO3 5, agar powder 20
斜面培养基(g/L)：甘油20，葡萄糖20，酵母粉5，琼脂粉20，CaCO310Slant medium (g/L): glycerol 20, glucose 20, yeast powder 5, agar powder 20, CaCO3 10
发酵培养基(g/L)：甘油60-140，酵母粉5，消泡剂0.1，MgSO4·7H2O 0.1，CaCO32Fermentation medium (g/L): glycerin 60-140, yeast powder 5, defoamer 0.1, MgSO4 7H2 O 0.1, CaCO3 2
将筛选的氧化葡萄糖酸杆菌野生株L-6和突变株I-2-239分别复苏后，在斜面于30℃活化，接种到种子培养液，培养到对数生长中后期后，分别接种于含有60g/L和120g/L甘油的灭菌发酵培养基中，培养基的初始pH为4.0～8.0，发酵温度是30～33℃，转速为200rpm。在室温下以7000～12000rpm离心15min后，得到菌体，在105℃下干燥至恒重后测定生物量，离心后的上清液用以DHA测定。野生株L-6发酵54h，DHA的平均产量为47.98g/L；突变株I-2-239发酵36h，DHA的平均产量为103.5g/L。尽管两者的生物量没有显著差别，但与野生株L-6比较，突变株I-2-239能够耐受高浓度的甘油，表现出生长速率加快，催化效率提高，发酵周期缩短。After recovering the screened Gluconobacter oxydans wild strain L-6 and mutant strain I-2-239 respectively, they were activated on the slope at 30°C, inoculated into the seed culture solution, cultivated to the middle and late stages of logarithmic growth, and then inoculated in the culture medium containing In the sterilized fermentation medium of 60g/L and 120g/L glycerol, the initial pH of the medium is 4.0-8.0, the fermentation temperature is 30-33°C, and the rotation speed is 200rpm. After centrifugation at 7000-12000 rpm at room temperature for 15 minutes, the bacterial cells were obtained, dried at 105° C. to constant weight, and the biomass was measured, and the centrifuged supernatant was used for DHA determination. The wild strain L-6 was fermented for 54 hours, and the average yield of DHA was 47.98g/L; the mutant strain I-2-239 was fermented for 36 hours, and the average yield of DHA was 103.5g/L. Although there was no significant difference in biomass between the two, compared with the wild strain L-6, the mutant strain I-2-239 could tolerate high concentrations of glycerol, exhibited faster growth rate, higher catalytic efficiency, and shorter fermentation period.
实施例2：野生株和突变株的酶活力测定比较Embodiment 2: the enzyme activity assay comparison of wild strain and mutant strain
实施例1中发酵液分别离心，获得的菌体细胞用pH 6.0的磷酸缓冲液清洗3次，按照100mg细胞：1mL磷酸缓冲液的比例重悬菌体，再加入含有100g/L甘油的发酵液中生物转化3h，离心菌体，测定上清液的DHA含量。甘油脱氢酶的酶活定义为每小时生成DHA的量。野生株和突变株的甘油脱氢酶的酶活力分别为0.19±0.05mM DHA/h和0.34±0.05mM DHA/h。平行比较，突变株的甘油脱氢酶活性比野生株明显提高。The fermentation broth in Example 1 was centrifuged separately, and the obtained bacterial cells were washed 3 times with a phosphate buffer solution of pH 6.0, and the bacterial cells were resuspended according to the ratio of 100 mg cells: 1 mL phosphate buffer solution, and then the fermentation broth containing 100 g/L glycerin was added After 3 hours of biotransformation, the cells were centrifuged, and the DHA content of the supernatant was determined. The enzyme activity of glycerol dehydrogenase is defined as the amount of DHA produced per hour. The enzyme activities of glycerol dehydrogenase in wild strain and mutant strain were 0.19±0.05mM DHA/h and 0.34±0.05mM DHA/h, respectively. In parallel comparison, the glycerol dehydrogenase activity of the mutant strain was significantly higher than that of the wild strain.
实施例3：野生株和突变株的甘油脱氢酶基因转录水平分析和比较Embodiment 3: analysis and comparison of the glycerol dehydrogenase gene transcription level of wild strain and mutant strain
分别选择对数生长后期的野生株和突变株菌体细胞，提取总RNA，反转录后以cDNA为模板，以管家基因16SrDNA为内参，进行荧光实时定量PCR，测定甘油脱氢酶基因sldA和甘油脱氢酶基因sldB的转录水平。突变株甘油脱氢酶基因sldA转录水平是野生株的4.8倍，而sldB转录水平达到了野生株的5.4倍。突变株甘油脱氢酶转录水平显著的提高，这将有助于实现甘油脱氢酶基因的高效表达，从而使得单位细胞的甘油脱氢酶催化活性得到显著的提高。The somatic cells of the wild strain and the mutant strain in the late logarithmic growth stage were selected respectively, and total RNA was extracted. After reverse transcription, cDNA was used as a template and the housekeeping gene 16SrDNA was used as an internal reference to perform fluorescence real-time quantitative PCR to determine the glycerol dehydrogenase gene sldA and Transcript levels of the glycerol dehydrogenase gene sldB. The transcription level of glycerol dehydrogenase gene sldA in the mutant strain was 4.8 times that of the wild strain, while the transcription level of sldB was 5.4 times that of the wild strain. The mutant glycerol dehydrogenase transcription level is significantly improved, which will help to realize the high-efficiency expression of the glycerol dehydrogenase gene, so that the catalytic activity of the glycerol dehydrogenase per unit cell is significantly improved.
实施例4：突变菌株中特定核苷酸序列甘油脱氢酶基因的重组载体构建和表达Embodiment 4: the recombinant vector construction and expression of specific nucleotide sequence glycerol dehydrogenase gene in the mutant strain
重组质粒构建：Recombinant plasmid construction:
以提取的氧化葡萄糖酸突变菌株基因组DNA为模板，以5’-CCCAAGCTTATGCCGAATACT-3’为上游引物，以5’-CCGGAATTCTCAGCCCTTGTG-3’为下游引物，进行PCR扩增，得到甘油脱氢酶基因(sldAB)全长，参照《分子克隆实验指南》(美国Sambrook等编写)第三版，将基因插入到pMD-19质粒中的HindIII和EcoRI酶切位点，转化进E.coli DH5α，挑取阳性转化子，酶切鉴定后，得到重组质粒pMD-sldAB，委托测序公司验证其序列的正确性。Using the extracted oxidative gluconic acid mutant strain genomic DNA as a template, 5'-CCCAAGCTT ATGCCGAATACT-3' as an upstream primer, and 5'-CCGGAATTC TCAGCCCTTGTG-3' as a downstream primer, perform PCR amplification to obtain glycerol dehydrogenation The full length of the enzyme gene (sldAB), referring to the third edition of "Molecular Cloning Experiment Guide" (written by Sambrook, etc., USA), inserted the gene into the HindIII and EcoRI restriction sites in the pMD-19 plasmid, and transformed it into E.coli DH5α, The positive transformants were picked and identified by enzyme digestion to obtain the recombinant plasmid pMD-sldAB, and a sequencing company was entrusted to verify the correctness of its sequence.
以上述突变菌株基因组DNA为模板，以5’-GCACATGTCGCGGATGTTCAGGTGTTC-3’(上游引物)和5’-GTCTTCTACCGGAGAGGACCCTTCT-3’(下游引物)为引物，PCR扩增pqq基因簇(带有pqqABCDE启动子和部分开放阅读框)，双酶切后，构建重组质粒pMD-pqq，委托测序公司测序验证其序列的正确性。Using the genomic DNA of the above mutant strain as a template, 5'-GCACATGTCGCGGATGTTCAGGTGTTC-3' (upstream primer) and 5'-GTCTTCTACCGGAGAGGACCCTTCT-3' (downstream primer) as primers, PCR amplified the pqq gene cluster (with the pqqABCDE promoter and part Open reading frame), after double enzyme digestion, construct the recombinant plasmid pMD-pqq, and entrust a sequencing company to perform sequencing to verify the correctness of its sequence.
再分别以测序正确的重组质粒pMD-sldAB和pMD-pqq为模板，为了表达甘油脱氢酶和PQQ合成酶，将sldAB和pqqABCDE克隆到pET28a的T7启动子下游的NdeI和EcoRI位点，生成pET28a-sldAB-pqq。Then, using the correctly sequenced recombinant plasmids pMD-sldAB and pMD-pqq as templates, in order to express glycerol dehydrogenase and PQQ synthetase, clone sldAB and pqqABCDE into the NdeI and EcoRI sites downstream of the T7 promoter of pET28a to generate pET28a -sldAB-pqq.
转化和表达：Transformation and expression:
采用氯化钙法将上述重组质粒pET28a-sldAB-pqq转入E.coli BL21中，在含有Amp抗性的LB平板上生长12-16h，挑选转化的阳性单克隆。The above-mentioned recombinant plasmid pET28a-sldAB-pqq was transferred into E.coli BL21 by the calcium chloride method, grown on the LB plate containing Amp resistance for 12-16 hours, and the transformed positive single clone was selected.
将含有pET28a-sldAB-pqq重组质粒的E.coli BL21工程菌按照1％的接种量接入含有100μg/mL Amp抗性、40～100g/L甘油的LB培养基，37℃，220rpm摇床中培养3～6h使OD600约为0.8～1.2时，加入IPTG至终浓度10～40mg/mL诱导，按照实施例1发酵转化，实现了重组菌转化甘油生成DHA。Inoculate the E.coli BL21 engineered bacteria containing the pET28a-sldAB-pqq recombinant plasmid into the LB medium containing 100μg/mL Amp resistance and 40-100g/L glycerol according to the inoculum amount of 1%, in a shaker at 37°C and 220rpm After culturing for 3-6 hours to make the OD600 about 0.8-1.2, add IPTG to a final concentration of 10-40 mg/mL to induce, and ferment and transform according to Example 1 to realize the transformation of glycerol into DHA by the recombinant bacteria.
实施例5：特定核苷酸序列甘油脱氢酶基因在氧化葡萄糖酸杆菌的表达Embodiment 5: the expression of specific nucleotide sequence glycerol dehydrogenase gene in Gluconobacter oxidans
参照分子克隆的方法，以实施例4中重组质粒pMD-sldAB为模板，扩增得到的sldAB基因的PCR产物，采用限制性内切酶HindIII和EcoRI分别对sldAB基因的PCR产物和pBBR1MCS-2质粒双酶切，用T4连接酶连接后生成pBBR-sldAB，重组质粒转进E.coli DH5α中，在含有50μg/mL的卡那霉素的LB培养基中初筛后测序验证sldAB序列的正确性。Referring to the method of molecular cloning, using the recombinant plasmid pMD-sldAB in Example 4 as a template, amplifying the PCR product of the sldAB gene obtained, using restriction endonucleases HindIII and EcoRI to treat the PCR product of the sldAB gene and the pBBR1MCS-2 plasmid respectively Double enzyme digestion, ligation with T4 ligase to generate pBBR-sldAB, the recombinant plasmid was transformed into E.coli DH5α, and the correctness of the sldAB sequence was verified by sequencing after preliminary screening in LB medium containing 50 μg/mL kanamycin .
从含有pBBR-sldAB的E.coli DH5α重组菌中提取重组质粒；制备G.oxydans感受态细胞；采用电转化方法，将重组质粒pBBR-sldAB转进制备好的G.oxydans感受态细胞中，加入甘油种子培养基培养2-4小时。再取100μL涂布在含有50μg/mL的卡那霉素的甘油琼脂平板中，在30℃恒温培养箱中培养，挑选饱满圆滑的菌落发酵。按照实施例1发酵转化，在140g/L甘油的发酵培养基中，最终获得DHA的产量为125.3g/L，实现了重组菌的甘油转化。Extract the recombinant plasmid from the E.coli DH5α recombinant bacteria containing pBBR-sldAB; prepare G.oxydans competent cells; use the electroporation method to transfer the recombinant plasmid pBBR-sldAB into the prepared G.oxydans competent cells, add Glycerol seed medium was incubated for 2-4 hours. Then take 100 μL and spread it on a glycerol agar plate containing 50 μg/mL kanamycin, culture it in a constant temperature incubator at 30°C, and select plump and smooth colonies for fermentation. According to the fermentation transformation of Example 1, in the fermentation medium of 140g/L glycerol, the yield of DHA finally obtained was 125.3g/L, and the glycerol transformation of the recombinant bacteria was realized.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611086184.3ACN108102941A (en) | 2016-11-25 | 2016-11-25 | The Gluconobacter oxvdans of one plant of glycerol dehydrogenase gene sequence of combination containing certain films and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611086184.3ACN108102941A (en) | 2016-11-25 | 2016-11-25 | The Gluconobacter oxvdans of one plant of glycerol dehydrogenase gene sequence of combination containing certain films and its application |
| Publication Number | Publication Date |
|---|---|
| CN108102941Atrue CN108102941A (en) | 2018-06-01 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611086184.3APendingCN108102941A (en) | 2016-11-25 | 2016-11-25 | The Gluconobacter oxvdans of one plant of glycerol dehydrogenase gene sequence of combination containing certain films and its application |
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| CN (1) | CN108102941A (en) |
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| CN115717132A (en)* | 2022-07-27 | 2023-02-28 | 中国科学院上海高等研究院 | Glycerol dehydrogenase mutant, engineering bacteria producing dihydroxyacetone and use thereof |
| CN118562844A (en)* | 2024-06-04 | 2024-08-30 | 杭州佳嘉乐生物技术有限公司 | A strain metabolic modification method for increasing dihydroxyacetone production |
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| JP2011147378A (en)* | 2010-01-20 | 2011-08-04 | National Institute Of Advanced Industrial Science & Technology | Method for producing dihydroxyacetone |
| CN101948878A (en)* | 2010-09-02 | 2011-01-19 | 南京工业大学 | Application of gluconobacter oxydans in preparation of 1, 3-dihydroxyacetone |
| CN102392056A (en)* | 2011-12-09 | 2012-03-28 | 华东理工大学 | Genetically engineered strain and method for producing dihydroxyacetone by using the same |
| CN103789250A (en)* | 2014-02-19 | 2014-05-14 | 天津实发中科百奥工业生物技术有限公司 | 1,3-dihydroxyacetone high-yielding strain and construction method thereof |
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| CN115717132A (en)* | 2022-07-27 | 2023-02-28 | 中国科学院上海高等研究院 | Glycerol dehydrogenase mutant, engineering bacteria producing dihydroxyacetone and use thereof |
| CN115717132B (en)* | 2022-07-27 | 2025-07-04 | 中国科学院上海高等研究院 | Glycerol dehydrogenase mutant, engineered bacteria producing dihydroxyacetone and uses thereof |
| CN118562844A (en)* | 2024-06-04 | 2024-08-30 | 杭州佳嘉乐生物技术有限公司 | A strain metabolic modification method for increasing dihydroxyacetone production |
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