WO2024130475A1 - Engineered bacterium for preventing and treating non-alcoholic steatohepatitis, construction method therefor, and use thereof - Google Patents
Engineered bacterium for preventing and treating non-alcoholic steatohepatitis, construction method therefor, and use thereof Download PDFInfo
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Definitions
- the present invention relates to an engineered probiotic and a construction method and application thereof, and more specifically, to an engineered E. coli Nissle 1917 that can synthesize polyphenol compounds with therapeutic activity and its related applications for preventing and/or treating non-alcoholic fatty liver disease.
- Nonalcoholic steatohepatitis is a fatty liver disease characterized by excess liver fat, liver inflammation, and fibrosis.
- NASH is a further development of nonalcoholic fatty liver disease (NAFLD) and includes varying degrees of liver damage. NASH is histologically different from simple fatty liver, in which only fat accumulation without inflammation and fibrosis is present.
- NAFLD nonalcoholic fatty liver disease
- the number of NAFLD cases is expected to increase from 83.1 million in 2015 (approximately 25% of the population) to 100.9 million in 2030. The proportion of NASH among these cases will increase from 20% to 27%.
- Polyphenol compounds extracted from plants have the characteristics of treating insulin resistance, promoting fatty acid ⁇ -oxidation, and inhibiting the expression of pro-inflammatory factors.
- polyphenol compounds can effectively treat non-alcoholic fatty liver disease, but the drug half-life of these polyphenol compounds is relatively short, generally within half an hour, which is very unfavorable for the treatment of chronic diseases.
- Existing studies have found that by adding polyphenol compounds (p-coumaric acid) to the feed of mice and eating them for a long time, the efflux of cholesterol can be effectively increased, and the lipid content of the central obese tissue of mice and the low-density lipoprotein in the blood can be reduced.
- this implementation scheme is not suitable for humans, because mice can restrict their diet for a long time, but it cannot be applied to humans.
- probiotics can enhance the density of the patient's intestinal barrier, reduce the toxins secreted by intestinal microorganisms entering the liver and further worsening its fibrosis; probiotics can secrete some anti-inflammatory molecules to reduce liver inflammatory factors; some probiotics (E. coli Nissle 1917) can significantly reduce the ammonia content in the blood of patients with liver disease, thereby achieving the effect of reducing liver fibrosis.
- One object of the present invention is to provide an engineered bacterium for preventing and treating non-alcoholic fatty hepatitis.
- Another object of the present invention is to provide a method for constructing the engineered bacteria.
- Another object of the present invention is to provide applications of the engineered bacteria.
- the present invention provides an engineered bacterium, which is an engineered bacterium obtained by genetically modifying intestinal microorganisms to produce active substances capable of preventing and/or treating non-alcoholic fatty liver disease.
- the intestinal microorganisms are intestinal probiotics.
- the intestinal microorganism is selected from any one of E. coli Nissle 1917, Bacillus subtilis, Lactobacillus, Lactococcus, Bacteroides, and Bifidobacterium.
- the active substance is a polyphenol compound.
- the polyphenolic compound is selected from one or more of p-coumaric acid, caffeic acid, ferulic acid, gallic acid, catechol, resveratrol, and naringenin;
- the polyphenolic compound is coumaric acid and/or caffeic acid.
- the polyphenolic compound is p-coumaric acid
- the engineered bacteria highly expresses tyrosine aminotransferase.
- the tyrosine aminotransferase is a tyrosine aminotransferase derived from Rhodotorula glutinosus. More preferably, the amino acid sequence of the tyrosine aminotransferase is as shown in SEQ ID NO:9.
- the engineered bacteria of the present invention also highly express one or more of aroG, aroL, tyrA, aroF, aroH, aroK, pheA, pheL, tktA, and ppsA.
- the present invention also provides a method for preparing the engineered bacteria (construction method), the method comprising:
- the gene of the active substance for preventing and/or treating non-alcoholic fatty liver disease is integrated into the genome of intestinal microorganisms, so that the obtained engineered bacteria can produce the active substance for preventing and/or treating non-alcoholic fatty liver disease.
- the corresponding gene string is integrated into the genome of the intestinal microorganism by the ⁇ -RED homologous recombination method.
- the gene is transcribed by the J23102 promoter.
- the present invention also provides the use of the engineered bacteria in the preparation of a drug for preventing and/or treating non-alcoholic fatty liver disease.
- the present invention also provides a drug for preventing and/or treating non-alcoholic fatty liver disease, which comprises: the engineered bacteria and/or its metabolites described in the present invention, and pharmaceutically acceptable excipients.
- the present invention also provides a method for preventing and/or treating non-alcoholic fatty liver disease, which comprises administering to a subject an effective amount of the engineered bacteria of the present invention and/or its metabolites, and/or the drug of the present invention.
- the present invention attempts to engineer the probiotic E. coli Nissle 1917 to enable it to efficiently utilize a variety of carbohydrates and synthesize polyphenol compounds, thereby achieving multiple therapeutic targets at the same time and achieving a more efficient NASH treatment effect.
- the present invention engineers the human intestinal probiotic E. coli Nissle 1917 so that it can continuously convert carbohydrates in the intestine and synthesize polyphenolic compounds with therapeutic activity (such as p-coumaric acid, caffeic acid, etc.). This can not only play the role of probiotics in enhancing the density of the intestinal barrier and reducing the blood ammonia concentration of NASH patients, but also continuously play the role of polyphenolic compounds in reducing pro-inflammatory factors, promoting lipid metabolism, improving insulin resistance and other multiple target effects. Since the polyphenolic compounds and intestinal probiotics involved in this system are harmless to the human body, it can be predicted that the live bacterial drug will not have adverse side effects on patients. Moreover, from the subsequent mouse experiments, the live bacterial drug does not have obvious side effects.
- the present invention is to engineer the probiotic Nissle 1917 so that it can utilize carbohydrates in the intestine and continuously synthesize p-CA (p-coumaric acid) with therapeutic effects, thereby achieving the purpose of sustained release.
- p-CA p-coumaric acid
- the side effects of the entire system are relatively low compared to currently clinically available drugs, such as obeticholic acid.
- the probiotic drug in the present invention mainly involves its genome modification, and the ultimate goal is to enhance the ability of chassis bacteria to synthesize p-coumaric acid. Thereby, the sustained release of p-CA in the intestine can be achieved.
- Previous studies have shown that p-CA can be obtained from tyrosine through the catalytic reaction of transaminase. For this reason, the present invention attempts to transform the EcN chassis from two aspects.
- the present invention attempts to integrate the corresponding gene string into the genome of EcN by the method of ⁇ -RED homologous recombination.
- all genes are transcribed by the J23102 promoter (sequence: ttgacagctagctcagtcctaggtactgtgctagc).
- RgTAL TAL derived from Rhodotorula glutinosus
- the p-CA of the transformed strain can be increased by 400%.
- the present invention provides an engineered Escherichia coli Nissle 1917, the genome of which carries related enzymes for highly expressing tyrosine and related enzymes for converting tyrosine into p-coumaric acid.
- the engineered Escherichia coli Nissle 1917 highly expresses RgTAL, or highly expresses RgTAL and tyrA, or highly expresses RgTAL, tyrA and aroL, or highly expresses RgTAL, tyrA, aroG and aroL.
- the engineered strain of the present invention has the ability to metabolize various carbohydrates such as glucose, fructose, galactose, maltose, sucrose, lactose, etc.
- the engineered strain of the present invention can be taken orally and treat non-alcoholic fatty hepatitis. Since p-coumaric acid also has the effects of anti-inflammatory, fat-reducing, and improving insulin resistance, oral administration of the engineered strain of the present invention also has the effects of anti-inflammatory, fat-reducing, and improving insulin resistance.
- the engineered bacteria of the present invention reside in the intestine for a long time and have the ability to metabolize various carbohydrates, so that the concentration of coumaric acid in the blood can be maintained at a high level for a long time, which plays a certain sustained-release effect and can solve the problems of short blood half-life and low effective utilization of coumaric acid to a certain extent.
- the present invention also provides the use of the engineered bacteria in producing p-coumaric acid. That is, the present invention provides a method for producing p-coumaric acid, which comprises: culturing the engineered bacteria of the present invention to produce p-coumaric acid. Specifically, the culturing can be carried out in M9 medium or LB medium.
- the carbon source in the culture medium may include one or more of glucose, fructose, galactose, maltose, sucrose, and lactose.
- Suitable culture conditions may be culturing at 37° C. for more than 0.5 hours, such as 0.5 hours to 3 days, preferably 0.5 hours to 2 days.
- the present invention provides an engineered bacterium for preventing and treating non-alcoholic fatty hepatitis, and a method for constructing and using the same.
- the present invention has better NASH treatment effects, including significant improvement in liver fibrosis, reduction in liver and blood lipids, and decrease in liver specific gravity.
- the invented drug also has the effect of reducing lipid toxicity in hepatocytes and maintaining the normal state of cells.
- the invented drug theoretically has no obvious side effects, and no obvious side effects were observed during the animal experiment when the invented drug was perfused.
- FIG1 is a schematic diagram of the engineered probiotics of the present invention achieving sustained release of p-CA and being used to treat NASH.
- Figure 2 is a schematic diagram of the design and modification of the EcN chassis cell.
- FIG. 4 shows the effect of different concentrations of glucose on the production of p-CA by the engineered bacteria of the present invention.
- FIG5 shows the yield of the product p-CA when different carbon sources are used as substrates.
- FIG6 is a growth curve diagram of EcN after knocking out dapA.
- FIG. 7 is a diagram showing the distribution of the engineered bacteria of the present invention in the mouse intestine over time.
- FIG8 is a graph showing the change of p-CA content in the serum of mice infused with the engineered probiotics of the present invention over time.
- FIG9 shows the experimental results of the engineered bacteria of the present invention in treating model mice induced by a high-fat diet.
- FIG. 10 is a staining image of liver sections of db/db mice induced by a high-fat diet.
- FIG. 11 is a picture of a Sirius Red liver section of a carbon tetrachloride-induced liver fibrosis model.
- FIG. 12 shows the results of analyzing the fiber area ratio in the stained liver sections.
- each raw reagent material can be obtained commercially, and the experimental method without specifying the specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
- the probiotic Nissle 1917 (purchase website: https://ambershield.com/product/mutaflor-capsules/) was engineered as follows: aroG, tyrA, aroL, TAL and other genes were inserted into multiple sites (malEK, LacZ, yicS/nepI, etc.) of the genome of E. coli Nissle 1917 by the ⁇ RED homologous recombination method, where the length of the homologous arm selected was 1000 bases, and the resistance gene used for screening was kana resistance, and its two segments were connected with FRT sequences, which can be used for subsequent elimination of resistance genes.
- the engineered probiotics can utilize carbohydrates in the intestine and continuously synthesize p-CA (p-coumaric acid) with therapeutic effects, thereby achieving the purpose of sustained release.
- p-CA p-coumaric acid
- the side effects of the entire system are relatively low compared to the drugs currently entering the clinic, such as obeticholic acid ( Figure 1).
- the present invention attempts to modify the EcN chassis from two aspects. On the one hand, by upregulating the conversion from glucose to tyrosine in bacteria, such as high expression of tyrA, aroG, aroL, etc.; on the other hand, by heterologously expressing tyrosine transaminase (TAL) with high catalytic activity in EcN to achieve the conversion from tyrosine to p-CA ( Figure 2).
- TAL tyrosine transaminase
- the present invention attempts to integrate the corresponding gene string into the genome of EcN by the method of ⁇ -RED homologous recombination.
- all genes are transcribed through the J23102 promoter (sequence is SEQ ID NO:1: ttgacagctagctcagtcctaggtactgtgctagc).
- RgTAL TAL derived from Rhodotorula glutinosus
- the present invention Compared with the NASH treatment drugs currently under development, the present invention has better NASH treatment effects, including significant improvement of liver fibrosis, reduction of liver and blood lipids, and reduction of liver specific gravity. From the results of liver section staining, the invented drug also has the effect of reducing lipid toxicity in liver cells and maintaining the normal state of cells. At the same time, the invented drug theoretically has no obvious side effects, and no obvious side effects were observed during the animal experiment when the invented drug was perfused.
- the present invention tests the amount of p-CA produced by the engineered probiotics when different carbon sources are used as substrates.
- carbohydrates involved in daily diet including three monosaccharides: glucose, fructose, and galactose, and three disaccharides: maltose, sucrose, and lactose.
- the efficiency of p-CA production of maltose and lactose is comparable to that of the three monosaccharides, but when the substrate is sucrose, the yield of p-CA is significantly reduced, and the p-CA detected in the supernatant after 3 hours is only 20 ⁇ M, which may be due to the lack of relevant pathways for sucrose metabolism in EcN cells.
- sucrose will be broken down into two monosaccharides, glucose and fructose, in the intestine, the bacteria can still metabolize sucrose in the end.
- the present invention also knocks out the dapA gene necessary for EcN growth, so that the possible unpredictable pollution problems caused by the engineered bacteria in the intestine and the environment can be effectively suppressed.
- Figure 6 experimental conditions: in a 96-well plate, 150 microliters of LB culture medium are added to each well, and then the corresponding overnight cultured modified strains are added to the corresponding wells at a volume ratio of 1:100, where the +DAP indicates that 100 micrograms/ml of diaminopimelic acid is added, and the -DAP indicates that diaminopimelic acid is not added
- the bacteria will only grow normally when DAP is added exogenously, and when there is no DAP, the bacteria cannot grow and replicate. There is not enough DAP in the intestine and the environment to allow the engineered bacteria to grow normally, so the biological hazards that may be caused by the bacteria can be effectively suppressed.
- mice fed with a high-fat diet were used as a model, and 5x10 10 CFU of the engineered strain were injected intragastricly to observe the liver fat, liver specific gravity and blood lipids of the mice.
- the results are shown in Figure 9. From the results, it can be seen that the experimental group can reduce the content of tristearin in the liver and the specific gravity of the liver compared with the control group, with significant differences. At the same time, the tristearin and cholesterol levels in the blood have decreased to a certain extent. This shows that the engineered strain has a positive therapeutic effect on the fatty acid metabolism of mice.
- liver cells in the control group all have obvious fat toxicity, with a large number of fat particles in the cells and damaged cell morphology. Relatively speaking, although there is also a certain amount of fat accumulation in the liver of the experimental group, there are still some relatively intact fat cells, and there is no excessive accumulation of fat in the cells. This result is similar to the above.
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Abstract
An engineered bacterium for preventing and treating non-alcoholic steatohepatitis, a construction method therefor, and use thereof. The present invention provides an engineered bacterium that is capable of generating active substances for preventing and/or treating non-alcoholic fatty liver by genetically modifying intestinal microorganisms. Particularly provided is an engineered bacterium E.coli Nissle 1917 capable of synthesizing polyphenolic compounds with therapeutic activity. Also provided are a construction method for the described engineered bacterium and associated use thereof in the treatment of non-alcoholic steatohepatitis.
Description
本发明是关于一种工程益生菌及其构建方法与应用，具体而言，是关于一种可以合成具有治疗活性的多酚类化合物的工程E.coli Nissle 1917及其用于预防和/或治疗非酒精性脂肪性肝炎的相关应用。The present invention relates to an engineered probiotic and a construction method and application thereof, and more specifically, to an engineered E. coli Nissle 1917 that can synthesize polyphenol compounds with therapeutic activity and its related applications for preventing and/or treating non-alcoholic fatty liver disease.
非酒精性脂肪性肝炎(NASH)是一种以肝脏脂肪过多、肝脏炎症和纤维化为特征的脂肪肝疾病。NASH属于非酒精性脂肪性肝病(NAFLD)进一步发展的结果，其包括不同程度的肝损伤。NASH在组织学上与简单的脂肪肝不同，后者仅存在脂肪堆积而没有炎症和纤维化。在美国，NAFLD病例的数量预计将从2015年的8310万(约占人口的25％)增加到2030年的1.009亿。这些病例中NASH的比例将从20％上升到27％。这种不断上升的疾病发生率将对社会生产带来越来越大的经济负担，并伴随着越来越多的肝硬化和终末期肝病患者的肝脏移植需求以及肝细胞癌的发生。与其他肝病的发病率相比，在NASH中出现的肝细胞癌比例大多(～35％-50％)发生在患者肝硬化和进行常规癌症筛查之前。因此，相比于有其他病因引起的肝细胞癌相比，这些肿瘤往往体积更大，且更难以进行治疗。在全球范围内，NAFLD的患病率估计约为25％，中东和南美最高，非洲最低。考虑到非酒精性脂肪肝的发病率和肥胖指数成正相关性以及全球肥胖人群的比率不断上升的情况，未来NASH药物的市场需求将会飞速上升。根据Evaluatepharma的预测，全球2025年NASH市场规模将达到600亿美元。Nonalcoholic steatohepatitis (NASH) is a fatty liver disease characterized by excess liver fat, liver inflammation, and fibrosis. NASH is a further development of nonalcoholic fatty liver disease (NAFLD) and includes varying degrees of liver damage. NASH is histologically different from simple fatty liver, in which only fat accumulation without inflammation and fibrosis is present. In the United States, the number of NAFLD cases is expected to increase from 83.1 million in 2015 (approximately 25% of the population) to 100.9 million in 2030. The proportion of NASH among these cases will increase from 20% to 27%. This rising incidence of the disease will impose an increasing economic burden on social production, accompanied by an increasing demand for liver transplants in patients with cirrhosis and end-stage liver disease, as well as the occurrence of hepatocellular carcinoma. Compared with the incidence of other liver diseases, the proportion of hepatocellular carcinomas that occur in NASH (~35%-50%) mostly occurs before patients have cirrhosis and undergo routine cancer screening. Therefore, compared with hepatocellular carcinomas caused by other etiologies, these tumors tend to be larger and more difficult to treat. Globally, the prevalence of NAFLD is estimated to be about 25%, with the highest prevalence in the Middle East and South America and the lowest in Africa. Considering the positive correlation between the incidence of non-alcoholic fatty liver disease and the obesity index and the increasing rate of obesity in the world, the market demand for NASH drugs will rise rapidly in the future. According to Evaluatepharma's forecast, the global NASH market size will reach US$60 billion in 2025.
尽管如此，目前尚没有一款被批准用于非酒精性脂肪性肝炎治疗的药物。尽管很多药物正在申请临床研究，但几乎所有药物都没能通过临床3期试验。其中比较有希望的一款药物是FXR激动剂的奥贝胆酸。尽管临床3期结果显示该药物可以有效缓解和逆转肝脏纤维化，但是其并不能有效降低患者的低密度脂蛋白含量，同时在其有效剂量下，大多患者出现瘙痒等不良反应，最终未能受到FDA的认可和批准。目前开发的药物大多针对于单一靶点，但是NASH的发病机制较为复杂，目前的研究发现NASH的发病机制涉及到胰岛素抵抗，肝脏脂肪酸堆积，肝脏促炎因子上升等，针对单一靶点难以取得有效的治疗效果也在意料之中。因此需要开发出一种新型的、能够同时针对多 个靶点的药物有望能够有效治疗NASH疾病。Despite this, there is currently no drug approved for the treatment of non-alcoholic fatty liver disease. Although many drugs are applying for clinical research, almost all of them have failed to pass the Phase 3 clinical trial. One of the more promising drugs is obeticholic acid, an FXR agonist. Although the Phase 3 clinical results showed that the drug can effectively alleviate and reverse liver fibrosis, it cannot effectively reduce the patient's low-density lipoprotein content. At the same time, at its effective dose, most patients experience adverse reactions such as itching, and ultimately failed to be recognized and approved by the FDA. Most of the drugs currently developed target a single target, but the pathogenesis of NASH is relatively complex. Current studies have found that the pathogenesis of NASH involves insulin resistance, liver fatty acid accumulation, and increased liver pro-inflammatory factors. It is also expected that it is difficult to achieve effective therapeutic effects against a single target. Therefore, it is necessary to develop a new type of drug that can target multiple targets at the same time, which is expected to effectively treat NASH.
植物中提取的多酚类化合物具有治疗胰岛素抵抗、促进脂肪酸β氧化以及抑制促炎因子的表达等特点，已有相关动物实验证明一些多酚类化合物可以有效治疗非酒精性脂肪肝，但是这些多酚类化合物的药物半衰期时间相对较短，一般在半小时以内，这对于慢性疾病的治疗非常不利。现有的研究发现通过在小鼠的饲料中添加多酚类化合物(对香豆酸)并长期食用，可以有效增加胆固醇的外排，并减少小鼠中心肥胖组织的脂质含量以及血液中的低密度脂蛋白。但是这一实施方案并不适用于人体，因为小鼠可以长时间限制其饮食，但是却不能应用于人。Polyphenol compounds extracted from plants have the characteristics of treating insulin resistance, promoting fatty acid β-oxidation, and inhibiting the expression of pro-inflammatory factors. There are related animal experiments that have shown that some polyphenol compounds can effectively treat non-alcoholic fatty liver disease, but the drug half-life of these polyphenol compounds is relatively short, generally within half an hour, which is very unfavorable for the treatment of chronic diseases. Existing studies have found that by adding polyphenol compounds (p-coumaric acid) to the feed of mice and eating them for a long time, the efflux of cholesterol can be effectively increased, and the lipid content of the central obese tissue of mice and the low-density lipoprotein in the blood can be reduced. However, this implementation scheme is not suitable for humans, because mice can restrict their diet for a long time, but it cannot be applied to humans.
同时，研究发现肠道微生物也会影响NASH的疾病进程，通过服用一些益生菌可以缓解NASH的症状。其原因在于：益生菌可以增强患者的肠道屏障致密性，减少肠道中的微生物分泌的毒素进入肝脏而使其纤维化进一步恶化；益生菌会分泌一些抗炎分子，减少肝脏炎症因子；有些益生菌(E.coli Nissle 1917)可以显著减少肝脏疾病患者血液中的氨含量，从而达到减少肝脏纤维化的效果。At the same time, studies have found that intestinal microorganisms can also affect the course of NASH, and taking some probiotics can alleviate the symptoms of NASH. The reasons are: probiotics can enhance the density of the patient's intestinal barrier, reduce the toxins secreted by intestinal microorganisms entering the liver and further worsening its fibrosis; probiotics can secrete some anti-inflammatory molecules to reduce liver inflammatory factors; some probiotics (E. coli Nissle 1917) can significantly reduce the ammonia content in the blood of patients with liver disease, thereby achieving the effect of reducing liver fibrosis.
现有治疗NASH的药物大多针对单一的治疗靶点，因此NASH的临床治疗效果比较差，而且很多药物有较大的的副作用。Most of the existing drugs for treating NASH target a single therapeutic target, so the clinical treatment effect of NASH is relatively poor, and many drugs have significant side effects.
发明内容Summary of the invention
本发明的一个目的在于提供一种防治非酒精性脂肪性肝炎的工程菌。One object of the present invention is to provide an engineered bacterium for preventing and treating non-alcoholic fatty hepatitis.
本发明的另一目的在于提供所述工程菌的构建方法。Another object of the present invention is to provide a method for constructing the engineered bacteria.
本发明的另一目的在于提供所述工程菌的应用。Another object of the present invention is to provide applications of the engineered bacteria.
一方面，本发明提供了一种工程菌，其是对肠道微生物进行基因改造而能够产生具有预防和/或治疗非酒精性脂肪肝的活性物质的工程菌。In one aspect, the present invention provides an engineered bacterium, which is an engineered bacterium obtained by genetically modifying intestinal microorganisms to produce active substances capable of preventing and/or treating non-alcoholic fatty liver disease.
根据本发明的具体实施方案，本发明所述的工程菌中，所述肠道微生物为肠道益生菌。According to a specific embodiment of the present invention, in the engineered bacteria of the present invention, the intestinal microorganisms are intestinal probiotics.
根据本发明的具体实施方案，本发明所述的工程菌中，所述肠道微生物选自E.coli Nissle 1917、枯草芽孢杆菌、乳酸杆菌、乳酸球菌、拟杆菌、双歧杆菌中的任意一种。According to a specific embodiment of the present invention, in the engineered bacteria described in the present invention, the intestinal microorganism is selected from any one of E. coli Nissle 1917, Bacillus subtilis, Lactobacillus, Lactococcus, Bacteroides, and Bifidobacterium.
根据本发明的具体实施方案，本发明所述的工程菌中，所述活性物质为多酚类化合物。According to a specific embodiment of the present invention, in the engineered bacteria of the present invention, the active substance is a polyphenol compound.
根据本发明的具体实施方案，本发明所述的工程菌中，所述多酚类化合物选自对 香豆酸、咖啡酸、阿魏酸、没食子酸、儿茶酚、白藜芦醇、柚皮素中的一种或多种；According to a specific embodiment of the present invention, in the engineered bacteria of the present invention, the polyphenolic compound is selected from one or more of p-coumaric acid, caffeic acid, ferulic acid, gallic acid, catechol, resveratrol, and naringenin;
根据本发明的具体实施方案，本发明所述的工程菌中，所述多酚类化合物为香豆酸和/或咖啡酸。According to a specific embodiment of the present invention, in the engineered bacteria of the present invention, the polyphenolic compound is coumaric acid and/or caffeic acid.
根据本发明的具体实施方案，本发明所述的工程菌中，所述多酚类化合物为对香豆酸，所述工程菌高表达酪氨酸转氨酶。优选地，所述酪氨酸转氨酶为来源于粘红酵母的酪氨酸转氨酶。更优选地，所述酪氨酸转氨酶的氨基酸序列如SEQ ID NO:9所示。According to a specific embodiment of the present invention, in the engineered bacteria of the present invention, the polyphenolic compound is p-coumaric acid, and the engineered bacteria highly expresses tyrosine aminotransferase. Preferably, the tyrosine aminotransferase is a tyrosine aminotransferase derived from Rhodotorula glutinosus. More preferably, the amino acid sequence of the tyrosine aminotransferase is as shown in SEQ ID NO:9.
根据本发明的具体实施方案，本发明所述的工程菌，其还高表达aroG、aroL、tyrA、aroF、aroH、aroK、pheA、pheL、tktA、ppsA中的一种或多种。According to a specific embodiment of the present invention, the engineered bacteria of the present invention also highly express one or more of aroG, aroL, tyrA, aroF, aroH, aroK, pheA, pheL, tktA, and ppsA.
另一方面，本发明还提供了所述的工程菌的制备方法(构建方法)，该方法包括：On the other hand, the present invention also provides a method for preparing the engineered bacteria (construction method), the method comprising:
将具有预防和/或治疗非酒精性脂肪肝的活性物质的基因整合到肠道微生物的基因组上，以使所得到的工程菌能够产生具有预防和/或治疗非酒精性脂肪肝的活性物质。The gene of the active substance for preventing and/or treating non-alcoholic fatty liver disease is integrated into the genome of intestinal microorganisms, so that the obtained engineered bacteria can produce the active substance for preventing and/or treating non-alcoholic fatty liver disease.
根据本发明的具体实施方案，本发明所述的工程菌的制备方法中，通过λ-RED同源重组的方法将相应的基因串整合到肠道微生物的基因组上。According to a specific embodiment of the present invention, in the method for preparing the engineered bacteria of the present invention, the corresponding gene string is integrated into the genome of the intestinal microorganism by the λ-RED homologous recombination method.
根据本发明的具体实施方案，本发明所述的工程菌的制备方法中，所述基因通过J23102启动子启动转录。According to a specific embodiment of the present invention, in the method for preparing the engineered bacteria of the present invention, the gene is transcribed by the J23102 promoter.
另一方面，本发明还提供了所述的工程菌在制备用于预防和/或治疗非酒精性脂肪肝的药物中的应用。On the other hand, the present invention also provides the use of the engineered bacteria in the preparation of a drug for preventing and/or treating non-alcoholic fatty liver disease.
另一方面，本发明还提供了一种预防和/或治疗非酒精性脂肪肝的药物，其包括：本发明所述的工程菌和/或其代谢产物，以及药学上可接受的辅料。On the other hand, the present invention also provides a drug for preventing and/or treating non-alcoholic fatty liver disease, which comprises: the engineered bacteria and/or its metabolites described in the present invention, and pharmaceutically acceptable excipients.
另一方面，本发明还提供了一种预防和/或治疗非酒精性脂肪肝的方法，该方法包括给予受试者有效量的本发明所述的工程菌和/或其代谢产物，和/或本发明所述的药物。On the other hand, the present invention also provides a method for preventing and/or treating non-alcoholic fatty liver disease, which comprises administering to a subject an effective amount of the engineered bacteria of the present invention and/or its metabolites, and/or the drug of the present invention.
根据本发明的一些具体实施方案，本发明尝试对益生菌E.coli Nissle 1917进行工程改造，使其能够高效利用多种碳水化合物并合成多酚类化合物，从而实现从多个治疗靶点同时入手，达到更高效的NASH治疗效果。According to some specific embodiments of the present invention, the present invention attempts to engineer the probiotic E. coli Nissle 1917 to enable it to efficiently utilize a variety of carbohydrates and synthesize polyphenol compounds, thereby achieving multiple therapeutic targets at the same time and achieving a more efficient NASH treatment effect.
本发明通过对人肠道益生菌E.coli Nissle 1917进行工程改造，使其在肠道中可以持续转化碳水化合物，并合成具有治疗活性的多酚类化合物(例如对香豆酸、咖啡酸等)，这样既能发挥益生菌增强肠道屏障致密性和减少NASH患者血液氨浓度的作用，同时也可以持续发挥多酚类化合物减少促炎因子、促进脂质代谢、改善胰岛素抵抗等多个靶点作用。由于这个体系中所涉及到的多酚类化合物和肠道益生菌都是对人体无害的， 因此可以预测该活菌药物不会对患者产生不好的副作用。而且从后续的小鼠实验来看，该活菌药物也确实不存在比较明显的副作用。The present invention engineers the human intestinal probiotic E. coli Nissle 1917 so that it can continuously convert carbohydrates in the intestine and synthesize polyphenolic compounds with therapeutic activity (such as p-coumaric acid, caffeic acid, etc.). This can not only play the role of probiotics in enhancing the density of the intestinal barrier and reducing the blood ammonia concentration of NASH patients, but also continuously play the role of polyphenolic compounds in reducing pro-inflammatory factors, promoting lipid metabolism, improving insulin resistance and other multiple target effects. Since the polyphenolic compounds and intestinal probiotics involved in this system are harmless to the human body, it can be predicted that the live bacterial drug will not have adverse side effects on patients. Moreover, from the subsequent mouse experiments, the live bacterial drug does not have obvious side effects.
本发明内容是对益生菌Nissle 1917进行工程改造，使其能够利用肠道中的碳水化合物，并不断合成具有治疗效果的p-CA(对香豆酸)，从而实现持续释放的目的。另外，由于p-CA分子和所使用的底盘细菌都是被认为是安全的，因此相比于目前进入临床的药物，如奥贝胆酸等，整个体系的副作用相对较低。The present invention is to engineer the probiotic Nissle 1917 so that it can utilize carbohydrates in the intestine and continuously synthesize p-CA (p-coumaric acid) with therapeutic effects, thereby achieving the purpose of sustained release. In addition, since the p-CA molecule and the chassis bacteria used are considered safe, the side effects of the entire system are relatively low compared to currently clinically available drugs, such as obeticholic acid.
本发明中的益生菌药物主要涉及其基因组改造，最终目的是为了提成底盘细菌合成对香豆酸的能力。从而可以实现p-CA在肠道中的持续释放。以往的研究表明，p-CA可以从酪氨酸经过转氨酶的催化反应得到。为此本发明尝试从两个方面对EcN底盘进行改造。一方面通过上调细菌内从葡萄糖到酪氨酸的转化，比如高表达tyrA，arog，arol等；另一方面通过EcN异源表达具有高催化活性的酪氨酸转氨酶(TAL)实现从酪氨酸到p-CA的转化。The probiotic drug in the present invention mainly involves its genome modification, and the ultimate goal is to enhance the ability of chassis bacteria to synthesize p-coumaric acid. Thereby, the sustained release of p-CA in the intestine can be achieved. Previous studies have shown that p-CA can be obtained from tyrosine through the catalytic reaction of transaminase. For this reason, the present invention attempts to transform the EcN chassis from two aspects. On the one hand, by upregulating the conversion from glucose to tyrosine in bacteria, such as high expression of tyrA, arog, arol, etc.; on the other hand, by heterologously expressing tyrosine transaminase (TAL) with high catalytic activity through EcN to achieve the conversion from tyrosine to p-CA.
本发明尝试通过λ-RED同源重组的方法将相应的基因串整合到EcN的基因组上，为了提高各蛋白的表达量，所有的基因都通过J23102启动子(序列为：ttgacagctagctcagtcctaggtactgtgctagc)启动转录。通过测试发现只整合RgTAL(源于粘红酵母的TAL)产生的菌株只能产生少量的p-CA，可能的原因在于底物酪氨酸产生的量不足。为此，通过继续将表达产酪氨酸的几个酶tyrA，aroG，aroL等的基因串到基因组可以将改造后菌株的p-CA提高400％。The present invention attempts to integrate the corresponding gene string into the genome of EcN by the method of λ-RED homologous recombination. In order to increase the expression of each protein, all genes are transcribed by the J23102 promoter (sequence: ttgacagctagctcagtcctaggtactgtgctagc). Through testing, it was found that the strain produced by integrating only RgTAL (TAL derived from Rhodotorula glutinosus) can only produce a small amount of p-CA, which may be due to the insufficient amount of substrate tyrosine produced. Therefore, by continuing to string the genes of several enzymes such as tyrA, aroG, aroL, etc. that express tyrosine production into the genome, the p-CA of the transformed strain can be increased by 400%.
与此同时，通过将益生菌与不同浓度的葡萄糖底物混合，通过检测p-CA的产量随时间变化的关系来探索底物浓度对产p-CA的影响。结果发现，在工程菌浓度一定的情况下，0.02％、0.2％和2％的葡萄糖对于产p-CA的效率并没有影响。相对于0.02％的葡萄糖浓度而言，工程菌可以在1小时内将其全部反应掉，导致之后由于底物不足而没有进一步的p-CA的合成。基于此，可以得出结论：葡萄糖扩散和运输并不会对p-CA的合成速率产生影响。At the same time, by mixing probiotics with different concentrations of glucose substrates, the effect of substrate concentration on p-CA production was explored by detecting the relationship between p-CA production and time. The results showed that when the concentration of engineered bacteria was constant, 0.02%, 0.2% and 2% glucose had no effect on the efficiency of p-CA production. Relative to the glucose concentration of 0.02%, the engineered bacteria can react all of it within 1 hour, resulting in no further synthesis of p-CA due to insufficient substrate. Based on this, it can be concluded that glucose diffusion and transport do not affect the synthesis rate of p-CA.
根据本发明的具体实施方案，本发明提供了一种工程的大肠杆菌Nissle 1917，基因组上携带高表达酪氨酸的相关酶以及将酪氨酸转化为对香豆酸的相关酶。具体而言，所述工程的大肠杆菌Nissle 1917高表达RgTAL，或者高表达RgTAL和tyrA，或者高表达RgTAL、tyrA和aroL，或者高表达RgTAL、tyrA、aroG和aroL。本发明的工程菌株具有可以代谢各种碳水化合物例如葡萄糖、果糖、半乳糖、麦芽糖、蔗糖、乳糖等的能力。 本发明的工程菌株可以通过口服并治疗非酒精性脂肪性肝炎。由于对香豆酸还具有消炎、减脂、改善胰岛素抵抗等效果，从而口服本发明的工程菌株同时具有消炎、减脂、改善胰岛素抵抗等效果。并且，本发明的工程菌在肠道中驻留较长时间以及具有可以代谢各种碳水化合物的能力，可以实现将香豆酸在血液中的浓度长时间维持在较高的水平，起到一定的缓释作用，可以一定程度上解决香豆酸血药半衰期短、有效利用度低的问题。According to a specific embodiment of the present invention, the present invention provides an engineered Escherichia coli Nissle 1917, the genome of which carries related enzymes for highly expressing tyrosine and related enzymes for converting tyrosine into p-coumaric acid. Specifically, the engineered Escherichia coli Nissle 1917 highly expresses RgTAL, or highly expresses RgTAL and tyrA, or highly expresses RgTAL, tyrA and aroL, or highly expresses RgTAL, tyrA, aroG and aroL. The engineered strain of the present invention has the ability to metabolize various carbohydrates such as glucose, fructose, galactose, maltose, sucrose, lactose, etc. The engineered strain of the present invention can be taken orally and treat non-alcoholic fatty hepatitis. Since p-coumaric acid also has the effects of anti-inflammatory, fat-reducing, and improving insulin resistance, oral administration of the engineered strain of the present invention also has the effects of anti-inflammatory, fat-reducing, and improving insulin resistance. In addition, the engineered bacteria of the present invention reside in the intestine for a long time and have the ability to metabolize various carbohydrates, so that the concentration of coumaric acid in the blood can be maintained at a high level for a long time, which plays a certain sustained-release effect and can solve the problems of short blood half-life and low effective utilization of coumaric acid to a certain extent.
另一方面，本发明还提供了所述的工程菌在生产对香豆酸中的应用。即本发明提供了一种生产对香豆酸的方法，该方法包括：培养本发明的工程菌，使其产生对香豆酸。具体地，可在M9培养基或LB培养基中进行培养。On the other hand, the present invention also provides the use of the engineered bacteria in producing p-coumaric acid. That is, the present invention provides a method for producing p-coumaric acid, which comprises: culturing the engineered bacteria of the present invention to produce p-coumaric acid. Specifically, the culturing can be carried out in M9 medium or LB medium.
具体地，所述培养基中的碳源可以包括葡萄糖、果糖、半乳糖、麦芽糖、蔗糖、乳糖中的一种或多种。适宜的培养条件可以是37℃培养0.5小时以上，例如0.5小时至3天，优选为0.5小时至2天。Specifically, the carbon source in the culture medium may include one or more of glucose, fructose, galactose, maltose, sucrose, and lactose. Suitable culture conditions may be culturing at 37° C. for more than 0.5 hours, such as 0.5 hours to 3 days, preferably 0.5 hours to 2 days.
整体而言，本发明提供了一种防治非酒精性脂肪性肝炎的工程菌及其构建方法与应用。相比于目前正在研发的治疗NASH药物，本发明具有更好的NASH治疗效果，包括显著的肝脏纤维化改善、肝脏和血液脂质的降低、肝脏比重的下降等。从肝脏切片染色结果来看，该发明药物还具有降低肝细胞中的脂质毒性，维持细胞的正常状态。与此同时该发明药物的从理论上来说不具有明显的副作用，在动物实验过程中灌注该发明药物也未观察到明显的副作用。In general, the present invention provides an engineered bacterium for preventing and treating non-alcoholic fatty hepatitis, and a method for constructing and using the same. Compared with the NASH treatment drugs currently under development, the present invention has better NASH treatment effects, including significant improvement in liver fibrosis, reduction in liver and blood lipids, and decrease in liver specific gravity. Judging from the results of liver section staining, the invented drug also has the effect of reducing lipid toxicity in hepatocytes and maintaining the normal state of cells. At the same time, the invented drug theoretically has no obvious side effects, and no obvious side effects were observed during the animal experiment when the invented drug was perfused.
图1为本发明的工程益生菌实现p-CA的持续释放并用于治疗NASH的示意图。FIG1 is a schematic diagram of the engineered probiotics of the present invention achieving sustained release of p-CA and being used to treat NASH.
图2为EcN底盘细胞设计改造示意图。Figure 2 is a schematic diagram of the design and modification of the EcN chassis cell.
图3.整合不同基因后的底盘菌株产p-CA的产量(RgTAL表示源于粘红酵母的TAL酶)。Figure 3. p-CA production by chassis strains after integration of different genes (RgTAL indicates TAL enzyme derived from Rhodotorula glutinosus).
图4显示不同浓度的葡萄糖对本发明的工程菌产p-CA的影响。FIG. 4 shows the effect of different concentrations of glucose on the production of p-CA by the engineered bacteria of the present invention.
图5显示不同碳源作为底物时产物p-CA的产量。FIG5 shows the yield of the product p-CA when different carbon sources are used as substrates.
图6为EcN敲除dapA后的生长曲线图。FIG6 is a growth curve diagram of EcN after knocking out dapA.
图7为本发明的工程菌在小鼠肠道中随时间的分布图。FIG. 7 is a diagram showing the distribution of the engineered bacteria of the present invention in the mouse intestine over time.
图8为灌注本发明的工程益生菌的小鼠血清中p-CA含量随时间变化图。FIG8 is a graph showing the change of p-CA content in the serum of mice infused with the engineered probiotics of the present invention over time.
图9显示本发明的工程菌治疗高脂饮食诱导的模型鼠的实验结果。FIG9 shows the experimental results of the engineered bacteria of the present invention in treating model mice induced by a high-fat diet.
图10为高脂饮食诱导的db/db鼠的肝脏切片染色图。FIG. 10 is a staining image of liver sections of db/db mice induced by a high-fat diet.
图11为四氯化碳诱导的肝脏纤维模型的天狼星红肝脏切片图。FIG. 11 is a picture of a Sirius Red liver section of a carbon tetrachloride-induced liver fibrosis model.
图12显示肝脏切片染色图中纤维面积占比分析结果。FIG. 12 shows the results of analyzing the fiber area ratio in the stained liver sections.
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的使用范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail in conjunction with specific embodiments, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of use of the present invention. In the embodiments, each raw reagent material can be obtained commercially, and the experimental method without specifying the specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。Unless specifically defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the relevant art.
实施例1Example 1
对益生菌Nissle 1917(采购网页：https://ambershield.com/product/mutaflor-capsules/)进行工程改造，方法如下：通过λRED同源重组的方法，分别在E.coli Nissle 1917的基因组多个位点(malEK，LacZ，yicS/nepI等)插入aroG、tyrA、aroL、TAL等多个基因，其中选择的同源臂长度为1000个碱基，用于筛选的抗性基因是卡纳抗性，其两段连有FRT序列，可以用于后续的抗性基因的消除。工程后的益生菌能够利用肠道中的碳水化合物，并不断合成具有治疗效果的p-CA(对香豆酸)，从而实现持续释放的目的。另外，由于p-CA分子和所使用的底盘细菌都是被认为是安全的，因此相比于目前进入临床的药物，如奥贝胆酸等，整个体系的副作用相对较低(图1)。The probiotic Nissle 1917 (purchase website: https://ambershield.com/product/mutaflor-capsules/) was engineered as follows: aroG, tyrA, aroL, TAL and other genes were inserted into multiple sites (malEK, LacZ, yicS/nepI, etc.) of the genome of E. coli Nissle 1917 by the λRED homologous recombination method, where the length of the homologous arm selected was 1000 bases, and the resistance gene used for screening was kana resistance, and its two segments were connected with FRT sequences, which can be used for subsequent elimination of resistance genes. The engineered probiotics can utilize carbohydrates in the intestine and continuously synthesize p-CA (p-coumaric acid) with therapeutic effects, thereby achieving the purpose of sustained release. In addition, since the p-CA molecule and the chassis bacteria used are considered safe, the side effects of the entire system are relatively low compared to the drugs currently entering the clinic, such as obeticholic acid (Figure 1).
以往的研究表明，p-CA可以从酪氨酸经过转氨酶的催化反应得到。为此本发明尝试从两个方面对EcN底盘进行改造。一方面通过上调细菌内从葡萄糖到酪氨酸的转化，比如高表达tyrA、aroG、aroL等；另一方面通过EcN异源表达具有高催化活性的酪氨酸转氨酶(TAL)实现从酪氨酸到p-CA的转化(图2)。Previous studies have shown that p-CA can be obtained from tyrosine through the catalytic reaction of transaminases. To this end, the present invention attempts to modify the EcN chassis from two aspects. On the one hand, by upregulating the conversion from glucose to tyrosine in bacteria, such as high expression of tyrA, aroG, aroL, etc.; on the other hand, by heterologously expressing tyrosine transaminase (TAL) with high catalytic activity in EcN to achieve the conversion from tyrosine to p-CA (Figure 2).
本发明尝试通过λ-RED同源重组的方法将相应的基因串整合到EcN的基因组上，为了提高各蛋白的表达量，所有的基因都通过J23102启动子(序列为SEQ ID NO:1：ttgacagctagctcagtcctaggtactgtgctagc)启动转录。通过测试发现只整合RgTAL(源于粘红酵母的TAL)产生的菌株只能产生少量的p-CA。通过继续将表达产酪氨酸的几个酶tyrA、 aroG、aroL等的基因串到基因组可以将改造后菌株的p-CA提高400％(图3，图中2×RgTAL tyrA aroL aroG表示这个菌的量增加了一倍)。其中下表各个蛋白的氨基酸序列及编码蛋白的基因序列如表1所示：The present invention attempts to integrate the corresponding gene string into the genome of EcN by the method of λ-RED homologous recombination. In order to increase the expression level of each protein, all genes are transcribed through the J23102 promoter (sequence is SEQ ID NO:1: ttgacagctagctcagtcctaggtactgtgctagc). Through testing, it was found that the strain produced by integrating only RgTAL (TAL derived from Rhodotorula glutinosus) can only produce a small amount of p-CA. By continuing to string the genes of several tyrosine-producing enzymes tyrA, aroG, aroL, etc. into the genome, the p-CA of the modified strain can be increased by 400% (Figure 3, 2×RgTAL tyrA aroL aroG in the figure means that the amount of this bacterium has doubled). The amino acid sequences of each protein in the following table and the gene sequences encoding the proteins are shown in Table 1:
表1Table 1
与此同时，通过将益生菌与含有不同浓度的葡萄糖的M9培养液混合，在37度摇床、220rpm培养，通过检测p-CA的产量随时间变化的关系来探索底物浓度对产p-CA的影响。从图4的结果可以发现，在2x10
10CFU工程菌与不同葡萄糖初始浓度的M9溶液混合，当葡萄糖浓度充足的时候，不同葡萄糖浓度对于产p-CA的效率并没有影响。相对于0.02％的葡萄糖浓度而言，工程菌可以在1小时内将其全部反应掉，导致之后由于底物不足而没有进一步的p-CA的合成。基于此，可以得出结论：葡萄糖扩散和运输并不会对p-CA的合成速率产生影响。
At the same time, by mixing probiotics with M9 culture medium containing different concentrations of glucose, culturing in a 37-degree shaker at 220 rpm, and detecting the relationship between the production of p-CA and time, the effect of substrate concentration on p-CA production was explored. From the results in Figure 4, it can be found that when 2x10 10 CFU of engineered bacteria were mixed with M9 solutions with different initial glucose concentrations, when the glucose concentration was sufficient, different glucose concentrations had no effect on the efficiency of p-CA production. Relative to a glucose concentration of 0.02%, the engineered bacteria can react all of it within 1 hour, resulting in no further synthesis of p-CA due to insufficient substrate. Based on this, it can be concluded that glucose diffusion and transport do not affect the synthesis rate of p-CA.
本发明相比于目前正在研发的治疗NASH药物，本发明具有更好的NASH治疗效果，包括显著的肝脏纤维化改善、肝脏和血液脂质的降低、肝脏比重的下降等。从肝脏切片染色结果来看，该发明药物还具有降低肝细胞中的脂质毒性，维持细胞的正常状态。与此同时该发明药物的从理论上来说不具有明显的副作用，在动物实验过程中灌注该发明药物也未观察到明显的副作用。Compared with the NASH treatment drugs currently under development, the present invention has better NASH treatment effects, including significant improvement of liver fibrosis, reduction of liver and blood lipids, and reduction of liver specific gravity. From the results of liver section staining, the invented drug also has the effect of reducing lipid toxicity in liver cells and maintaining the normal state of cells. At the same time, the invented drug theoretically has no obvious side effects, and no obvious side effects were observed during the animal experiment when the invented drug was perfused.
实施例2Example 2
工程菌催化香豆酸合成的体外表征In vitro characterization of coumaric acid synthesis catalyzed by engineered bacteria
在构建完底盘益生菌之后，本发明测试了工程益生菌对于不同的碳源作为底物的时候产p-CA的量。After constructing the chassis probiotics, the present invention tests the amount of p-CA produced by the engineered probiotics when different carbon sources are used as substrates.
本发明中，选取了六种平时饮食中会涉及到的碳水化合物，包括三种单糖：葡萄糖、果糖、半乳糖，以及三种双糖：麦芽糖、蔗糖、乳糖。In the present invention, six kinds of carbohydrates involved in daily diet are selected, including three monosaccharides: glucose, fructose, and galactose, and three disaccharides: maltose, sucrose, and lactose.
通过图5(实验条件：将37度100mL LB培养基过夜培养的工程菌株离心并获得菌体，用PBS清洗后离心并重悬到5mL PBS中，然后在50mL离心管加入10mL灭过菌的M9培养基中，2x10
10CFU的工程菌株溶液以及20％质量浓度各种糖溶液(最终浓度为0.2％)，然后将混合溶液在37度摇床、220rpm培养，分别在不同的时间点取样测溶液上清中的p-CA浓度)可以发现，对三种单糖而言，该工程细菌都能快速催化产生p-CA，反应速率接近160μM/h。对于三种双糖而言，麦芽糖和乳糖的产p-CA的效率和三种单糖相当，但是当底物是蔗糖时，p-CA的产率明显降低，3小时候上清检测到的p-CA只有20μM，可能的原因是EcN胞内缺少蔗糖代谢的相关途径。但是由于考虑到在肠道中，蔗糖会被分解成葡萄糖和果糖这两种单糖，因此从最终结果上该细菌依然是可以代谢掉蔗糖的。
From Figure 5 (experimental conditions: the engineered strain cultured overnight in 100 mL LB medium at 37 degrees was centrifuged to obtain the bacterial cells, washed with PBS, centrifuged and resuspended in 5 mL PBS, then added to 10 mL sterilized M9 medium, 2x10 10 CFU of the engineered strain solution and 20% mass concentration of various sugar solutions (final concentration of 0.2%) in a 50 mL centrifuge tube, and then the mixed solution was cultured in a 37 degree shaker at 220 rpm, and the p-CA concentration in the supernatant of the solution was measured at different time points) it can be found that for the three monosaccharides, the engineered bacteria can quickly catalyze the production of p-CA, and the reaction rate is close to 160 μM/h. For the three disaccharides, the efficiency of p-CA production of maltose and lactose is comparable to that of the three monosaccharides, but when the substrate is sucrose, the yield of p-CA is significantly reduced, and the p-CA detected in the supernatant after 3 hours is only 20 μM, which may be due to the lack of relevant pathways for sucrose metabolism in EcN cells. However, considering that sucrose will be broken down into two monosaccharides, glucose and fructose, in the intestine, the bacteria can still metabolize sucrose in the end.
考虑到实际使用的情况中的生物安全问题，本发明也将EcN生长必须的dapA基因敲除，这样就可以有效抑制工程细菌在肠道和环境中造成的可能的不可预测的污染问题。图6(实验条件：在96孔板中，每个孔分别加入150微升LB培养基，然后以1:100的体积比分别将对应的过夜培养的改造菌株加入到相应的孔中，其中标注+DAP的表示另外添加了100微克/毫升的二氨基庚二酸，标注-DAP则表示没有添加二氨基庚二酸)中可以看到细菌只有在外源添加DAP的情况下才会正常生长，而当没有DAP的情况下，细菌无法生长复制。而在肠道和环境中均没有足量的DAP可以使得工程细菌正常生长，因此可以有效抑制细菌可能引起的生物危害。Taking into account the biosafety issues in actual use, the present invention also knocks out the dapA gene necessary for EcN growth, so that the possible unpredictable pollution problems caused by the engineered bacteria in the intestine and the environment can be effectively suppressed. Figure 6 (experimental conditions: in a 96-well plate, 150 microliters of LB culture medium are added to each well, and then the corresponding overnight cultured modified strains are added to the corresponding wells at a volume ratio of 1:100, where the +DAP indicates that 100 micrograms/ml of diaminopimelic acid is added, and the -DAP indicates that diaminopimelic acid is not added) can be seen that the bacteria will only grow normally when DAP is added exogenously, and when there is no DAP, the bacteria cannot grow and replicate. There is not enough DAP in the intestine and the environment to allow the engineered bacteria to grow normally, so the biological hazards that may be caused by the bacteria can be effectively suppressed.
工程益生菌小鼠体内药代动力学Pharmacokinetics of engineered probiotics in mice
本实施例探索该工程益生菌在小鼠体内的药代动力学。通过给小鼠灌胃注射10
10CFU的工程菌之后，分别在0.5h、1h、3h、12h、24h和48h取小鼠肠道不同位置(胃，空肠，回肠，结肠，盲肠)的肠道内容物，然后进行涂板计数。结果如图7所示。从图中可以观察到小鼠小肠中工程EcN菌株在12h的时候还有较多的剩余。到24h的时候细菌主要存留在大肠位置。到48h时，肠道所有位置都检测不到工程细菌。
This example explores the pharmacokinetics of the engineered probiotics in mice. After gavage of 10 10 CFU of engineered bacteria to mice, the intestinal contents of different positions of the mouse intestine (stomach, jejunum, ileum, colon, cecum) were taken at 0.5h, 1h, 3h, 12h, 24h and 48h, and then plate counts were performed. The results are shown in Figure 7. It can be observed from the figure that there are still a lot of engineered EcN strains in the small intestine of mice at 12h. By 24h, the bacteria mainly remain in the large intestine. By 48h, no engineered bacteria were detected in all positions of the intestine.
将不同量的工程菌灌注到小鼠体内，然后检测不同时间点小鼠血清中的p-CA含量。从图8可以观察到直接灌注p-CA组，血清中的浓度快速达到峰值，但是在12h的时候几乎检测不到p-CA的残余，可见p-CA已经被代谢并排除体外。相对而言，灌注5×10
10CFU工程菌组在12h的时候血清中依然有1.5μg/mL的浓度。这一结果符合预期。
Different amounts of engineered bacteria were infused into mice, and then the p-CA content in the mouse serum was detected at different time points. As can be seen from Figure 8, in the direct infusion p-CA group, the concentration in the serum quickly reached a peak, but at 12 hours, almost no p-CA residue was detected, indicating that p-CA had been metabolized and excreted from the body. In contrast, the serum concentration of the 5×10 10 CFU engineered bacteria group was still 1.5 μg/mL at 12 hours. This result was in line with expectations.
工程益生菌减少模型鼠的脂质Engineered probiotics reduce lipids in model mice
本实施例以高脂饮食饲养的db/db鼠作为模型，通过灌胃注射5x10
10CFU工程菌株观察小鼠的肝脏脂肪、肝脏比重以及血脂情况。结果如图9。从结果可以得知实验组相比于对照组，可以降低肝脏的三硬脂酸甘油酯的含量以及肝脏的比重，具有显著性差异。同时，血液中的三硬脂酸甘油酯和胆固醇含量都有一定程度的下降。由此可见工程菌株对于小鼠脂肪酸代谢方面具有积极的治疗效果。
In this embodiment, db/db mice fed with a high-fat diet were used as a model, and 5x10 10 CFU of the engineered strain were injected intragastricly to observe the liver fat, liver specific gravity and blood lipids of the mice. The results are shown in Figure 9. From the results, it can be seen that the experimental group can reduce the content of tristearin in the liver and the specific gravity of the liver compared with the control group, with significant differences. At the same time, the tristearin and cholesterol levels in the blood have decreased to a certain extent. This shows that the engineered strain has a positive therapeutic effect on the fatty acid metabolism of mice.
同时，从图10的肝脏切片染色结果可以观察到，对照组的肝脏细胞均有明显的脂肪毒性，细胞内含有大量的脂肪粒，细胞形态出现损害。相对而言，实验组的肝脏中虽然也有一定的脂肪堆积，但是还是有一些脂肪细胞比较完整，胞内并没有出现脂肪过度聚集的情况，这一结果和上述类似。At the same time, from the results of liver section staining in Figure 10, it can be observed that the liver cells in the control group all have obvious fat toxicity, with a large number of fat particles in the cells and damaged cell morphology. Relatively speaking, although there is also a certain amount of fat accumulation in the liver of the experimental group, there are still some relatively intact fat cells, and there is no excessive accumulation of fat in the cells. This result is similar to the above.
工程益生菌缓解小鼠纤维化Engineered probiotics alleviate fibrosis in mice
本实施例在四氯化碳诱导小鼠肝脏纤维化的模型上验证工程菌的治疗效果。图11、图12结果可以观察到工程菌治疗组可以有效降低肝脏的纤维化程度，并具有显著性差异，其效果与目前临床使用的奥贝胆酸的效果相当。但是，结果发现未工程的EcN在纤维化抑制中也有一定的效果，可能的原因是发现EcN在临床上可以有效减少血浆中的氨的含量，而之前的小鼠实验发现血液中氨的减少可以有效抑制肝脏的纤维化程度。This example verifies the therapeutic effect of engineered bacteria in a model of liver fibrosis induced by carbon tetrachloride in mice. The results of Figures 11 and 12 show that the engineered bacteria treatment group can effectively reduce the degree of liver fibrosis, and there is a significant difference, which is equivalent to the effect of obeticholic acid currently used in clinical practice. However, the results show that unengineered EcN also has a certain effect in inhibiting fibrosis. The possible reason is that EcN has been found to effectively reduce the ammonia content in plasma in clinical practice, and previous mouse experiments have found that the reduction of ammonia in the blood can effectively inhibit the degree of liver fibrosis.
Claims (12)
- 一种工程菌，其是对肠道微生物进行基因改造而能够产生具有预防和/或治疗非酒精性脂肪肝的活性物质的工程菌。An engineered bacterium is an engineered bacterium that is produced by genetically modifying intestinal microorganisms to produce active substances that can prevent and/or treat non-alcoholic fatty liver disease.
- 根据权利要求1所述的工程菌，其中，所述肠道微生物为肠道益生菌。The engineered bacteria according to claim 1, wherein the intestinal microorganisms are intestinal probiotics.
- 根据权利要求1或2所述的工程菌，其中，所述肠道微生物选自E.coli Nissle 1917、枯草芽孢杆菌、乳酸杆菌、乳酸球菌、拟杆菌、双歧杆菌中的任意一种。The engineered bacteria according to claim 1 or 2, wherein the intestinal microorganism is selected from any one of E. coli Nissle 1917, Bacillus subtilis, Lactobacillus, Lactococcus, Bacteroides, and Bifidobacterium.
- 根据权利要求1所述的工程菌，其中，所述活性物质为多酚类化合物。The engineered bacteria according to claim 1, wherein the active substance is a polyphenol compound.
- 根据权利要求4所述的工程菌，其中，所述多酚类化合物选自对香豆酸、咖啡酸、阿魏酸、没食子酸、儿茶酚、白藜芦醇、柚皮素中的一种或多种；The engineered bacteria according to claim 4, wherein the polyphenolic compound is selected from one or more of p-coumaric acid, caffeic acid, ferulic acid, gallic acid, catechol, resveratrol, and naringenin;优选地，所述多酚类化合物为香豆酸和/或咖啡酸。Preferably, the polyphenol compound is coumaric acid and/or caffeic acid.
- 根据权利要求5所述的工程菌，其中，所述多酚类化合物为对香豆酸，所述工程菌高表达酪氨酸转氨酶；The engineered bacteria according to claim 5, wherein the polyphenol compound is p-coumaric acid, and the engineered bacteria highly expresses tyrosine aminotransferase;优选地，所述酪氨酸转氨酶为来源于粘红酵母的酪氨酸转氨酶；Preferably, the tyrosine aminotransferase is a tyrosine aminotransferase derived from Rhodotorula glutinosus;更优选地，所述酪氨酸转氨酶的氨基酸序列如SEQ ID NO:9所示。More preferably, the amino acid sequence of the tyrosine aminotransferase is as shown in SEQ ID NO:9.
- 根据权利要求5或6所述的工程菌，其还高表达aroG、aroL、tyrA、aroF、aroH、aroK、pheA、pheL、tktA、ppsA中的一种或多种。The engineered bacteria according to claim 5 or 6, further highly expresses one or more of aroG, aroL, tyrA, aroF, aroH, aroK, pheA, pheL, tktA, and ppsA.
- 权利要求1-7任一项所述的工程菌的制备方法，该方法包括：The method for preparing the engineered bacteria according to any one of claims 1 to 7, comprising:将具有预防和/或治疗非酒精性脂肪肝的活性物质的基因整合到肠道微生物的基因组上，以使所得到的工程菌能够产生具有预防和/或治疗非酒精性脂肪肝的活性物质；Integrating genes of active substances for preventing and/or treating non-alcoholic fatty liver disease into the genome of intestinal microorganisms, so that the resulting engineered bacteria can produce active substances for preventing and/or treating non-alcoholic fatty liver disease;优选地，通过λ-RED同源重组的方法将相应的基因串整合到肠道微生物的基因组上；Preferably, the corresponding gene string is integrated into the genome of the intestinal microorganism by the λ-RED homologous recombination method;更优选地，所述基因通过J23102启动子启动转录。More preferably, the gene is transcribed via the J23102 promoter.
- 权利要求1-7任一项所述的工程菌在生产对香豆酸中的应用；Use of the engineered bacteria according to any one of claims 1 to 7 in the production of p-coumaric acid;优选地，所述应用包括：培养所述的工程菌，使其产生对香豆酸；Preferably, the application comprises: culturing the engineered bacteria to produce p-coumaric acid;更优选地，培养基中的碳源包括葡萄糖、果糖、半乳糖、麦芽糖、蔗糖、乳糖中的一种或多种。More preferably, the carbon source in the culture medium includes one or more of glucose, fructose, galactose, maltose, sucrose and lactose.
- 权利要求1-7任一项所述的工程菌在制备用于预防和/或治疗非酒精性脂肪肝的药物中的应用。Use of the engineered bacteria according to any one of claims 1 to 7 in the preparation of a medicament for preventing and/or treating non-alcoholic fatty liver disease.
- 一种预防和/或治疗非酒精性脂肪肝的药物，其包括：权利要求1-7任一项所述的工程菌和/或其代谢产物，以及药学上可接受的辅料。A drug for preventing and/or treating non-alcoholic fatty liver disease, comprising: the engineered bacteria and/or its metabolites according to any one of claims 1 to 7, and pharmaceutically acceptable excipients.
- 一种预防和/或治疗非酒精性脂肪肝的方法，该方法包括给予受试者有效量的权利要求1-7任一项所述的工程菌和/或其代谢产物，和/或权利要求11所述的药物。A method for preventing and/or treating non-alcoholic fatty liver disease, comprising administering to a subject an effective amount of the engineered bacteria and/or its metabolites according to any one of claims 1 to 7, and/or the drug according to claim 11.
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