CN114921502A - Method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters - Google Patents

Method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters Download PDF

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CN114921502A
CN114921502A CN202210426003.6A CN202210426003A CN114921502A CN 114921502 A CN114921502 A CN 114921502A CN 202210426003 A CN202210426003 A CN 202210426003A CN 114921502 A CN114921502 A CN 114921502A
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李乃强
毕吻吻
陈辰
王学利
俞建勇
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Abstract

本发明涉及一种基于微生物生理参数进行反馈调控氮源流加的戊二酸生产方法,将菌种培养液接种于含有发酵培养基的发酵罐中,以发酵过程中的实时微生物生理参数氧气消耗速率(OUR)、二氧化碳生成速率(CER)为依据反馈调控氮源流加速度,发酵获得戊二酸。本发明产酸高,转化率高,容易规模化放大,为生物法戊二酸的产业化奠定基础。

Figure 202210426003

The invention relates to a glutaric acid production method based on microbial physiological parameters for feedback regulation of nitrogen source flow addition. A bacterial culture liquid is inoculated into a fermentation tank containing a fermentation medium, and the oxygen consumption rate of the real-time microbial physiological parameters in the fermentation process is used. (OUR) and carbon dioxide generation rate (CER) are based on feedback regulation of nitrogen source flow acceleration, and glutaric acid is obtained by fermentation. The invention has high acid production, high conversion rate, easy scale amplification, and lays a foundation for the industrialization of biological glutaric acid.

Figure 202210426003

Description

一种基于微生物生理参数进行反馈调控氮源流加的戊二酸生 产方法A method for the production of glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition

技术领域technical field

本发明属于生物领域,特别涉及一种基于微生物生理参数反馈调控氮源流加的戊二酸生产方法。The invention belongs to the field of biology, and particularly relates to a method for producing glutaric acid based on the feedback regulation of nitrogen source flow addition based on microbial physiological parameters.

背景技术Background technique

戊二酸可以合成戊二酸酐,广泛应用于橡胶、医药等领域。此外,戊二酸与不同的二元胺聚合可以得到新型的聚酰胺。目前,市场上的戊二酸大多是通过分离己二酸的副产物或者通过化学反应得到。化学合成戊二酸需要高温、高压和昂贵的催化剂,如通过丁内酯与剧毒化合物氰化钾开环,然后进行水解反应可以合成戊二酸,该工艺毒性大、成本高、工艺安全性低、环境不友好。通过分离己二酸的副产物来得到高纯度戊二酸需要多步结晶、收率低、成本高,限制了戊二酸的进一步应用。近年来,通过代谢工程改造微生物生产戊二酸成为研究的热点,包括大肠杆菌和谷氨酸棒杆菌在内的微生物被广泛开发合成戊二酸。5-氨基戊酸途径(AMV)途径被以为是最有指望的戊二酸生物合成途径。如图1所示,AMV途径是通过选择合适的宿主,如大肠杆菌或者谷氨酸棒杆菌,在其中过表达来源于恶臭假单胞菌(Pseudomonas putida)的赖氨酸2-单加氧酶(DavB)和δ-氨基戊酰胺酶(DavA),将赖氨酸代谢为5-氨基戊酸,再进一步通过5-氨基戊酸氨基转移酶(GabT)和琥珀酸半醛脱氢酶(GabD)得到戊二酸。迄今为止,生物法合成戊二酸的研究主要集中于对微生物的代谢工程改造,而对戊二酸的发酵工艺研究甚少。工业生产L-赖氨酸的微生物生产需要大量的氮源,如硫酸铵和液氨,而基于L-赖氨酸分解代谢的戊二酸生产需要逐步的脱氨和转氨反应。因此,氮源流加策略会干扰L-赖氨酸和戊二酸的合成,然而,没有相关文献报道氮源如何影响戊二酸的合成。同样如图1所示,L-赖氨酸到5-氨基戊酰胺需要氧气并产生二氧化碳,这与实时的微生物摄氧率(OUR)、二氧化碳释放率(CER)生理参数有关。本发明中,我们开发了一种基于实时的微生物生理参数进行反馈调节的氮源流加策略,并应用于戊二酸的生物法生产。该工艺产酸高,糖酸转化率高,容易放大到产业化规模。Glutaric acid can synthesize glutaric anhydride, which is widely used in rubber, medicine and other fields. In addition, the polymerization of glutaric acid with different diamines can give new polyamides. At present, glutaric acid on the market is mostly obtained by separating by-products of adipic acid or by chemical reaction. Chemical synthesis of glutaric acid requires high temperature, high pressure and expensive catalysts. For example, glutaric acid can be synthesized through the ring opening of butyrolactone and the highly toxic compound potassium cyanide, followed by hydrolysis. This process is toxic, costly and safe. Low and unfriendly environment. Obtaining high-purity glutaric acid by separating the by-product of adipic acid requires multi-step crystallization, low yield and high cost, which limits the further application of glutaric acid. In recent years, the production of glutaric acid by metabolically engineered microorganisms has become a research hotspot, and microorganisms including Escherichia coli and Corynebacterium glutamicum have been widely developed to synthesize glutaric acid. The 5-aminovaleric acid pathway (AMV) pathway is considered to be the most promising glutaric acid biosynthetic pathway. As shown in Figure 1, the AMV pathway is achieved by selecting a suitable host, such as Escherichia coli or Corynebacterium glutamicum, in which the lysine 2-monooxygenase derived from Pseudomonas putida is overexpressed (DavB) and δ-aminovaleramidase (DavA), which metabolize lysine to 5-aminovaleric acid, which is further processed by 5-aminovaleric acid aminotransferase (GabT) and succinate semialdehyde dehydrogenase (GabD ) to obtain glutaric acid. So far, the research on biosynthesis of glutaric acid has mainly focused on the metabolic engineering of microorganisms, while the fermentation process of glutaric acid has been rarely studied. Microbial production for industrial L-lysine production requires a large amount of nitrogen sources, such as ammonium sulfate and liquid ammonia, whereas glutaric acid production based on L-lysine catabolism requires stepwise deamination and transamination reactions. Therefore, the nitrogen source fed-addition strategy would interfere with the synthesis of L-lysine and glutaric acid, however, there is no relevant literature on how nitrogen sources affect the synthesis of glutaric acid. Also shown in Figure 1, L-lysine to 5-aminopentanamide requires oxygen and produces carbon dioxide, which is related to the real-time microbial oxygen uptake rate (OUR) and carbon dioxide release rate (CER) physiological parameters. In the present invention, we developed a nitrogen source fed-feed strategy based on real-time microbial physiological parameters for feedback regulation, and applied it to the biological production of glutaric acid. The process has high acid production, high sugar-acid conversion rate, and is easy to scale to industrial scale.

发明内容SUMMARY OF THE INVENTION

针对现有技术的缺陷,本发明所要解决的技术问题是提供一种基于微生物生理参数进行反馈调控氮源流加的戊二酸生产方法。Aiming at the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for producing glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition.

本发明的一种基于微生物生理参数进行反馈调控氮源流加的戊二酸生产方法,包括:A method for producing glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition, comprising:

将菌种接种于含有发酵培养基的发酵罐内,以实时的微生物生理参数为依据反馈调控氮源流加,进行发酵获得戊二酸;其中,微生物生理参数包括氧气消耗速率(OUR)、二氧化碳生成速率(CER)。The strain is inoculated into a fermenter containing a fermentation medium, and based on real-time microbial physiological parameters, the nitrogen source is fed back and regulated, and glutaric acid is obtained by fermentation; wherein, the microbial physiological parameters include oxygen consumption rate (OUR), carbon dioxide production rate rate (CER).

进一步地,将戊二酸生产菌培养液接种于含有发酵培养基的发酵罐内,在合适的培养基和培养条件下,微生物利用培养基生长,当实时的OUR、CER逐步上升到140–180m molL-1h-1,微生物进入戊二酸快速合成阶段,在该时,启动氮源流加。Further, the glutaric acid-producing bacteria culture solution was inoculated into a fermentor containing a fermentation medium. Under suitable medium and culture conditions, the microorganisms used the medium to grow. When the real-time OUR and CER gradually increased to 140–180 m. molL -1 h -1 , the microorganisms enter the stage of rapid synthesis of glutaric acid, and at this time, the nitrogen source feed is started.

所述调控氮源流加为调控氮源流加的速度,氮源流加的速度可以基于实时的OUR、CER自动反馈调节,也可以根据实时的OUR、CER手动调节。The said regulating nitrogen source flow is to regulate the speed of nitrogen source flow, and the speed of nitrogen source flow can be adjusted automatically based on real-time OUR and CER feedback, or can be manually adjusted according to real-time OUR and CER.

进一步地,当产酸期的OUR、CER低于140–180m mol L-1h-1,提高氮源流加速度,使得OUR、CER保持在140–180m mol L-1h-1,更优选为160–180m mol L-1h-1Further, when the OUR and CER in the acid production period are lower than 140-180 mmol L -1 h -1 , the nitrogen source flow acceleration is increased, so that the OUR and CER are kept at 140-180 mmol L -1 h -1 , more preferably 160 -180 mmol L -1 h -1 .

所述氮源为氨水、液氨、硫酸铵溶液、氯化铵溶液、尿素溶液、碳酸铵、碳酸氢铵溶液中的一种或几种,优选为硫酸铵溶液,其中,所述的溶液为水溶液。The nitrogen source is one or more of ammonia water, liquid ammonia, ammonium sulfate solution, ammonium chloride solution, urea solution, ammonium carbonate and ammonium bicarbonate solution, preferably ammonium sulfate solution, wherein the solution is aqueous solution.

当用氨水进行氮源流加时,可以同时用氨水来调控发酵液的pH值,使得pH值保持在6.7–7.2,也可以用氢氧化钠溶液来调控发酵液的pH值,氨水的浓度一般在25–28%(w/v),氢氧化钠溶液的浓度一般在5–40%(w/v)。When ammonia water is used for nitrogen source flow addition, ammonia water can be used to control the pH value of the fermentation broth at the same time, so that the pH value is kept at 6.7-7.2, and sodium hydroxide solution can also be used to control the pH value of the fermentation broth. The concentration of ammonia water is generally in 25–28% (w/v), the concentration of sodium hydroxide solution is generally 5–40% (w/v).

进一步优选地,流加10–50%(w/v)的硫酸铵溶液提供氮源,5–40%(w/v)氢氧化钠溶液调控发酵液的pH值为6.7–7.2。Further preferably, 10-50% (w/v) ammonium sulfate solution is fed to provide the nitrogen source, and 5-40% (w/v) sodium hydroxide solution is used to adjust the pH value of the fermentation broth to 6.7-7.2.

所述发酵过程中还包括流加葡萄糖溶液,使得发酵液中葡萄糖的浓度为0.5–1%(w/v)。所述流加葡萄糖溶液,其中葡萄糖溶液的浓度为50–70%(w/v)。The fermentation process also includes feeding glucose solution, so that the concentration of glucose in the fermentation broth is 0.5-1% (w/v). The glucose solution is fed in, wherein the concentration of the glucose solution is 50-70% (w/v).

所述发酵工艺参数包括:接种量10–20%,发酵温度35–37℃,控制发酵液pH值为6.7–7.2,搅拌转速和溶氧(DO)关联,使得DO维持在20–40%,罐压保持在0.05–0.1MPa,发酵周期为50–70h。The fermentation process parameters include: the inoculum size of 10-20%, the fermentation temperature of 35-37°C, the pH value of the fermentation broth is controlled to be 6.7-7.2, the stirring speed is related to dissolved oxygen (DO), so that the DO is maintained at 20-40%, The tank pressure was maintained at 0.05–0.1 MPa, and the fermentation period was 50–70 h.

所述发酵采用的培养基为:磷酸二氢钾3–8g/L,1–3g/L硫酸镁、0.01–0.03g/L硫酸亚铁、0.01–0.04g/L硫酸锰、5–15g/L硫酸铵、20–40g/L葡萄糖、5–30g/L玉米浆,0.5–3g/L甜菜碱,0.002–0.006g/L维生素B1、培养基中添加抗生素,其中所述抗生素为30–200μg/mL氨苄青霉素、30–100μg/mL氯霉素、30–200μg/mL卡那霉素的一种或者几种。The culture medium used in the fermentation is: potassium dihydrogen phosphate 3-8g/L, 1-3g/L magnesium sulfate, 0.01-0.03g/L ferrous sulfate, 0.01-0.04g/L manganese sulfate, 5-15g/L L ammonium sulfate, 20–40 g/L glucose, 5–30 g/L corn steep liquor, 0.5–3 g/L betaine, 0.002–0.006 g/L vitamin B1, antibiotics were added to the medium, wherein the antibiotics were 30–200 μg One or more of ampicillin/mL ampicillin, 30–100 μg/mL chloramphenicol, and 30–200 μg/mL kanamycin.

所述菌种为代谢工程改造的大肠杆菌或谷氨酸棒杆菌。The strain is metabolically engineered Escherichia coli or Corynebacterium glutamicum.

注:本发明中的质量体积百分比%(w/v)均为g/100ml。Note: The mass volume percent (w/v) in the present invention is all g/100ml.

进一步地,本实验采用的戊二酸生产菌为代谢工程菌株。戊二酸代谢工程菌的相关研究报道较多。基于AMV途径的戊二酸生产菌的改造,主要通过将含有来自于恶臭假单胞菌的的赖氨酸2-单加氧酶(DavB)编码基因和δ-氨基戊酰胺酶(DavA)编码基因的质粒转入宿主菌,并进一步通过质粒过表达5-氨基戊酸氨基转移酶(GabT)和琥珀酸半醛脱氢酶(GabD,得到戊二酸。赖氨酸是合成戊二酸的前体,因此增强赖氨酸的代谢流也是常用的手段。目前,戊二酸生产菌大多为改造的大肠杆菌或者谷氨酸棒杆菌。如Han等人的研究报告Glutaric acid production by systems metabolic engineering of an L-lysine–overproducing Corynebacterium glutamicum(2020,Pnas.117,30328-30334),Kim等人的研究报告Metabolic engineering of Corynebacterium glutamicum for theproduction of glutaric acid,a C5 dicarboxylic acid platformchemical(2019,Metab.Eng.51,99–109),Li等人的研究报告Targeting metabolic driving andintermediate influx in L-lysine catabolism for high-level glutarateproduction.(2019,Nat.Commun.10,3337),Rohles等人的研究报告Systems metabolicengineering of Corynebacterium glutamicum for the production of the carbon-5platform chemicals 5-aminovalerate and glutarate.(2016,Microb.Cell.Fact.15,1-13),Adkins等人的研究报告Engineering Escherichia coli for RenewableProduction of the 5-Carbon Polyamide Building-Blocks 5-Aminovalerate andGlutarate(2013,Biotechnol.Bioeng.110,1726-1734)。Further, the glutaric acid-producing bacteria used in this experiment were metabolically engineered strains. There are many related research reports on glutaric acid metabolism engineering bacteria. The transformation of glutaric acid-producing bacteria based on the AMV pathway, mainly by encoding lysine 2-monooxygenase (DavB) and δ-aminovaleramidase (DavA) genes from Pseudomonas putida The plasmid of the gene is transferred into the host bacteria, and 5-aminovaleric acid aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD) are further overexpressed through the plasmid to obtain glutaric acid. Lysine is used to synthesize glutaric acid. Therefore, enhancing the metabolic flow of lysine is also a commonly used method. At present, most of the glutaric acid producing bacteria are modified Escherichia coli or Corynebacterium glutamicum. Such as the research report of Han et al. Glutaric acid production by systems metabolic engineering of an L-lysine–overproducing Corynebacterium glutamicum (2020, Pnas.117, 30328-30334), Kim et al. Metabolic engineering of Corynebacterium glutamicum for the production of glutaric acid, a C5 dicarboxylic acid platformchemical (2019, Metab.Eng. 51,99–109), the research report of Li et al. Targeting metabolic driving and intermediate influx in L-lysine catabolism for high-level glutarate production. (2019, Nat. Commun. 10, 3337), the research report of Rohles et al. Systems metabolicengineering of Corynebacterium glutamicum for the production of the carbon-5platform chemicals 5-aminovalerate and glutarate. (2016, Microb. Cell. Fact. 15, 1-13), a study by Adkins et al. Engineering Escherichia coli for RenewableProduction of the 5-Carbon Polyamide Building-Blocks 5-Aminovalerate and Glutarate (2013, Biotechnol. Bioeng. 11 0, 1726-1734).

进一步地,以本发明人所在实验室基于AMV途径参考上述文献构建的代谢工程菌株Escherichia coli LQ-1合成戊二酸为例来阐述该过程,权利要求不限于本实验室菌种、相关培养基和培养方法。Further, this process is described with reference to the metabolic engineering strain Escherichia coli LQ-1 synthesizing glutaric acid constructed by the laboratory of the present inventor based on the AMV approach with reference to the above-mentioned document, and the claims are not limited to this laboratory strain, relevant culture medium and cultivation methods.

生物法戊二酸发酵包括甘油管种子活化、摇瓶种子培养、种子罐种子培养和发酵过程。Bioprocess glutaric acid fermentation includes glycerol tube seed activation, shake flask seed culture, seed tank seed culture and fermentation process.

甘油管自然解冻后,用消毒后的枪头取300uL于平板培养基上,尽量铺开,37℃恒温培养箱倒置培养24h,平板培养基为:4–10g/L磷酸二氢钾、0.3–1g/L七水硫酸镁、2–6g/L硫酸铵、1–5g/L酵母粉、5–10g/L蛋白胨、1–5g/L蔗糖,添加合适的抗生素,如30–200μg/mL氨苄青霉素、30–100μg/mL氯霉素、30–200μg/mL卡那霉素的一种或者几种。抗生素种类的添加取决于代谢工程菌株的抗生素抗性特征。平板上菌落形成菌苔,用接种环取整个平板菌体于种子摇瓶中,种子摇瓶培养基同平板培养基,37℃,170rpm摇床培养7h后,接种一级种子罐。After the glycerol tube was thawed naturally, use a sterilized pipette tip to take 300uL on the plate medium, spread it out as much as possible, and cultivate it upside down in a constant temperature incubator at 37°C for 24 hours. The plate medium is: 4-10g/L potassium dihydrogen phosphate, 0.3- 1g/L magnesium sulfate heptahydrate, 2–6g/L ammonium sulfate, 1–5g/L yeast powder, 5–10g/L peptone, 1–5g/L sucrose, add appropriate antibiotics such as 30–200μg/mL ampicillin One or more of penicillin, 30–100 μg/mL chloramphenicol, and 30–200 μg/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strains. The colony on the plate forms a bacterial moss, and the whole plate is taken with an inoculation loop and placed in a seed shaker flask.

一级种子罐的接种量0.1–1%,通风量0.4–0.6vvm,搅拌转速300–600rpm,罐压0.05–0.08MPa,发酵过程中控制DO>5%,发酵过程pH值控制为6.7–7.2,37℃,培养16–18h,OD600达到0.8–1.0(稀释25倍)后,接种于10L的种子罐中,种子罐培养基为4–10g/L磷酸二氢钾、0.3–1g/L七水硫酸镁、2–6g/L硫酸铵、1–5g/L酵母粉、5–10g/L蛋白胨、30–50g/L葡萄糖、30–100μg/mL氨苄青霉素、30–100μg/mL氯霉素。选择合适的抗生素,如30–200μg/mL氨苄青霉素、30–100μg/mL氯霉素、30–200μg/mL卡那霉素的一种或者几种。抗生素种类的添加取决于代谢工程菌株的抗生素抗性特征。The inoculation volume of the first-stage seed tank is 0.1-1%, the ventilation volume is 0.4-0.6vvm, the stirring speed is 300-600rpm, the tank pressure is 0.05-0.08MPa, the DO>5% is controlled during the fermentation process, and the pH value during the fermentation process is controlled at 6.7-7.2 , 37°C, cultured for 16–18h, OD 600 reached 0.8–1.0 (diluted 25 times), inoculated in a 10L seed pot, the seed pot medium was 4–10g/L potassium dihydrogen phosphate, 0.3–1g/L Magnesium sulfate heptahydrate, 2–6 g/L ammonium sulfate, 1–5 g/L yeast powder, 5–10 g/L peptone, 30–50 g/L glucose, 30–100 μg/mL ampicillin, 30–100 μg/mL chloramphenicol white. Choose an appropriate antibiotic, such as one or more of 30–200 μg/mL ampicillin, 30–100 μg/mL chloramphenicol, and 30–200 μg/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strains.

发酵工艺为接种量10–20%,通风量0.4–0.5vvm,搅拌转速300–800rpm,罐压0.05–0.1MPa,控制发酵过程中DO>20%,控制发酵过程pH值6.7–7.2,发酵过程中,流加50–70%的葡萄糖溶液,并控制发酵液残糖浓度在0.5–1%,发酵周期为50–70h。发酵培养基包括:磷酸二氢钾3–8g/L,1–3g/L硫酸镁、0.01–0.03g/L硫酸亚铁、0.01–0.04g/L硫酸锰、5–15g/L硫酸铵、20–40g/L葡萄糖、5–30g/L玉米浆,0.5–3g/L甜菜碱,0.002–0.006g/L维生素B1,选择合适的抗生素,如30–200μg/mL氨苄青霉素、30–100μg/mL氯霉素、30–200μg/mL卡那霉素的一种或者几种。抗生素种类的添加取决于代谢工程菌株的抗生素抗性特征。The fermentation process is 10-20% inoculum, 0.4-0.5vvm ventilation, 300-800rpm stirring speed, 0.05-0.1MPa tank pressure, DO>20% during fermentation, pH 6.7-7.2 during fermentation, fermentation process 50–70% glucose solution was added, and the residual sugar concentration of the fermentation broth was controlled at 0.5–1%, and the fermentation period was 50–70 h. Fermentation medium includes: potassium dihydrogen phosphate 3–8g/L, 1–3g/L magnesium sulfate, 0.01–0.03g/L ferrous sulfate, 0.01–0.04g/L manganese sulfate, 5–15g/L ammonium sulfate, 20–40g/L glucose, 5–30g/L corn steep liquor, 0.5–3g/L betaine, 0.002–0.006g/L vitamin B1, choose appropriate antibiotics, such as 30–200μg/mL ampicillin, 30–100μg/L One or more of chloramphenicol, 30–200 μg/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strains.

进一步地,采用气相色谱法测定发酵液中的戊二酸含量,具体为发酵液取样后,离心机5000rpm离心5分钟,取上清液进行测定。气相色谱仪为GC2010pro(SHIMADZU,Japan),自动进样器,进样量0.5μL,分流比20:1。进口温度290℃,载气流速1.2mL/min,,检测器FID设定温度320℃。色谱柱起始温度为180℃,维持2分钟,以每分钟15℃的速率升温至300℃,维持7分钟。色谱柱型号为Wondacap-5:30m◇0.25mm◇0.25μm。Further, gas chromatography was used to measure the glutaric acid content in the fermentation broth, specifically, after sampling the fermentation broth, centrifuge at 5000 rpm for 5 minutes, and take the supernatant for measurement. The gas chromatograph was GC2010pro (SHIMADZU, Japan), an autosampler, the injection volume was 0.5 μL, and the split ratio was 20:1. The inlet temperature was 290 °C, the carrier gas flow rate was 1.2 mL/min, and the detector FID setting temperature was 320 °C. The initial temperature of the chromatographic column was 180°C, maintained for 2 minutes, and the temperature was increased to 300°C at a rate of 15°C per minute and maintained for 7 minutes. The column model is Wondacap-5: 30m◇0.25mm◇0.25μm.

进一步地,菌体生物量用分光光度计测定600nm下的吸光值,采用OD600来表示。发酵液的氨氮浓度采用常规凯氏定氮法测定。发酵液中的中间体赖氨酸采用常规的茚三酮比色法来测定。Further, the bacterial biomass was measured with a spectrophotometer for absorbance at 600 nm, and expressed by OD 600 . The ammonia nitrogen concentration of the fermentation broth was determined by the conventional Kjeldahl method. The intermediate lysine in the fermentation broth was determined by a conventional ninhydrin colorimetric method.

进一步地,发酵过程中,利用在线尾气分析仪检测尾气中的二氧化碳和氧气含量,利用国强生化设备公司FUS-30L高级发酵罐所带的biostar软件在线采集DO、CER、OUR、RQ等过程参数。Further, during the fermentation process, the carbon dioxide and oxygen content in the exhaust gas was detected by an online exhaust gas analyzer, and the process parameters such as DO, CER, OUR, and RQ were collected online using the biostar software of the FUS-30L advanced fermentation tank of Guoqiang Biochemical Equipment Company. .

有益效果beneficial effect

本发明基于发酵过程中的在线微生物生理特性参数OUR、CER指导的氮源调控策略在生物法戊二酸中的应用,采用该方法戊二酸产量高、糖酸转化率高,容易进行规模化放大。The invention is based on the application of the nitrogen source regulation strategy guided by the on-line microbial physiological characteristic parameters OUR and CER in the fermentation process in the biological method of glutaric acid. The method has high glutaric acid yield, high sugar-acid conversion rate, and is easy to scale. enlarge.

附图说明Description of drawings

图1为基于AMV途径的戊二酸合成过程中的脱氨、脱羧反应。Figure 1 shows the deamination and decarboxylation reactions in the synthesis of glutaric acid based on the AMV pathway.

图2为仅流加25%氨水,控制氨水流加速度,使得产酸期OUR、CER在60–80m mol L- 1h-1的戊二酸实时发酵过程参数图。Figure 2 is a parameter diagram of the real-time fermentation process of glutaric acid in which 25% ammonia water is only added and the acceleration of ammonia water flow is controlled, so that OUR and CER are at 60–80 mmol L - 1 h -1 during acid production.

图3为用30%氢氧化钠溶液调控发酵液pH,控制25%氨水流加速度,使得产酸期OUR、CER在120–140m mol L-1h-1的戊二酸实时发酵过程参数图。Figure 3 is a parameter diagram of the real-time fermentation process of glutaric acid in the acid production period of OUR and CER at 120–140 mmol L -1 h -1 by adjusting the pH of the fermentation broth with 30% sodium hydroxide solution and controlling the acceleration of 25% ammonia water flow.

图4为流加50%硫酸铵溶液,控制硫酸铵流加速度,使得产酸前期OUR和CER保持在160–180m mol L-1h-1,产酸中后期OUR和CER保持在60–80m mol L-1h-1的戊二酸实时发酵过程参数图。Fig. 4 shows the flow rate of 50% ammonium sulfate solution, and the flow rate of ammonium sulfate is controlled, so that OUR and CER are kept at 160–180 mmol L -1 h -1 in the early stage of acid production, and 60–80 mmol L in the middle and late stage of acid production. Parameter diagram of real-time fermentation process of glutaric acid for L -1 h -1 .

图5为流加50%硫酸铵溶液,控制硫酸铵流加速度,使得产酸前期OUR和CER保持在140–160m mol L-1h-1,产酸中后期OUR和CER保持在80–100m mol L-1h-1的戊二酸实时发酵过程参数图。Figure 5 shows the flow rate of 50% ammonium sulfate solution, and the flow rate of ammonium sulfate is controlled, so that OUR and CER are kept at 140–160 mmol L -1 h -1 in the early stage of acid production, and 80–100 mmol in the middle and late stage of acid production. Parameter diagram of real-time fermentation process of glutaric acid for L -1 h -1 .

图6为流加50%硫酸铵溶液,控制硫酸铵流加速度,使得产酸期OUR、CER分别在60–80mmol L-1h-1和80–10m mol L-1h-1两种方案时的发酵过程戊二酸产酸速率曲线和菌体生长曲线图。Figure 6 shows the flow rate of adding 50% ammonium sulfate solution and controlling the flow rate of ammonium sulfate, so that the OUR and CER in the acid production period are respectively 60–80 mmol L -1 h -1 and 80–10 mmol L -1 h -1 in the two schemes The glutaric acid production rate curve and bacterial growth curve of the fermentation process.

图7为流加50%硫酸铵溶液,控制硫酸铵流加速度,使得产酸期到发酵结束的OUR、CER保持在160–180m mol L-1h-1的戊二酸实时发酵过程参数图。Figure 7 shows the parameters of the real-time fermentation process of glutaric acid with 50% ammonium sulfate solution added and the flow rate of ammonium sulfate controlled to keep OUR and CER at 160–180 mmol L -1 h -1 from the acid production period to the end of the fermentation.

具体实施方式Detailed ways

下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

实施例1Example 1

基于微生物生理参数OUR、CER进行反馈调控氨水流加速度Feedback regulation of ammonia flow acceleration based on microbial physiological parameters OUR and CER

采用本实验室构建的代谢工程菌株Escherichia coli LQ-1进行戊二酸发酵,构建方法参考文献用常规方法进行,主要通过将来自于恶臭假单胞菌的的赖氨酸2-单加氧酶(DavB)编码基因和δ-氨基戊酰胺酶(DavA)编码基因的质粒转入宿主菌,并进一步通过质粒过表达5-氨基戊酸氨基转移酶(GabT)和琥珀酸半醛脱氢酶(GabD。发酵具体工艺如下:The metabolic engineering strain Escherichia coli LQ-1 constructed in this laboratory was used for glutaric acid fermentation. The construction method was carried out by conventional methods, mainly by lysine 2-monooxygenase from Pseudomonas putida. (DavB) encoding gene and δ-aminovaleramidase (DavA) encoding gene were transformed into host bacteria, and further overexpressed 5-aminovaleric acid aminotransferase (GabT) and succinate semialdehyde dehydrogenase ( GabD. The specific process of fermentation is as follows:

(1)甘油管自然解冻后,用消毒后的枪头取300uL于平板培养基上,尽量铺开,37℃恒温培养箱倒置培养24h。活化培养基为:4g/L磷酸二氢钾、0.5g/L七水硫酸镁、4.5g/L硫酸铵、5g/L酵母粉、8g/L蛋白胨、3g/L蔗糖、50μg/mL氨苄青霉素、50μg/mL氯霉素。(1) After the glycerol tube was thawed naturally, use a sterilized pipette tip to take 300uL on the plate medium, spread it out as much as possible, and incubate it upside down in a constant temperature incubator at 37°C for 24 hours. The activation medium is: 4g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 4.5g/L ammonium sulfate, 5g/L yeast powder, 8g/L peptone, 3g/L sucrose, 50μg/mL ampicillin , 50μg/mL chloramphenicol.

(2)平板上菌落形成菌苔,用接种环取整个平板菌体于种子摇瓶中,37℃,170rpm摇床培养7h后,接种一级种子罐。摇瓶培养基配方同平板培养基。(2) The colony on the plate forms a bacterial moss, and the whole plate bacterial cells are taken into a seed shaker with an inoculation loop, and after culturing for 7 hours at 37° C., a 170 rpm shaker, inoculate a first-class seed tank. The shake flask medium formula is the same as the plate medium.

(3)种子罐工艺:接种量0.1%,通风量0.4vvm,搅拌转速300–600rpm,罐压0.05–0.08MPa,培养过程中控制DO>5%,25%的氨水控制发酵过程pH值6.7,37℃,培养16–18h,OD600达到0.8–1.0(稀释25倍)后,接种于30L发酵罐。一级种子罐培养基为:6g/L磷酸二氢钾、0.5g/L七水硫酸镁、3g/L硫酸铵、4g/L酵母粉、6g/L蛋白胨、40g/L葡萄糖、50μg/mL氨苄青霉素、50μg/mL氯霉素。(3) Seed tank process: the inoculation volume is 0.1%, the ventilation volume is 0.4vvm, the stirring speed is 300-600rpm, the tank pressure is 0.05-0.08MPa, the DO>5% is controlled during the cultivation process, and the pH value of the fermentation process is controlled by 25% ammonia water to 6.7, After culturing for 16–18 h at 37°C, the OD 600 reached 0.8–1.0 (diluted 25 times), and then inoculated into a 30L fermenter. The first-class seed tank medium is: 6g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 3g/L ammonium sulfate, 4g/L yeast powder, 6g/L peptone, 40g/L glucose, 50μg/mL Ampicillin, 50 μg/mL chloramphenicol.

(4)发酵工艺:接种量14%,搅拌转速和DO关联,使得发酵过程中DO20%–40%,罐压0.05–0.1MPa,通风量0.4–0.6vvm,控制发酵温度为37℃,发酵过程pH值维持在6.7,发酵过程中,流加70%的葡萄糖溶液,并控制发酵液残糖浓度在0.5–1%,发酵周期为60h。发酵培养基:1.6g/L七水硫酸镁、0.03g/L七水硫酸亚铁、0.032g/L一水硫酸锰、10g/L硫酸铵、磷酸二氢钾5g/L、30g/L葡萄糖、25g/L玉米浆,2.2g/L甜菜碱,0.006g/L维生素B1、50μg/mL氨苄青霉素、50μg/mL氯霉素。(4) Fermentation process: the inoculum amount is 14%, the stirring speed is related to DO, so that during the fermentation process DO is 20%-40%, the tank pressure is 0.05-0.1MPa, the ventilation volume is 0.4-0.6vvm, and the fermentation temperature is controlled at 37 °C. The fermentation process The pH value was maintained at 6.7. During the fermentation process, 70% glucose solution was added, and the residual sugar concentration of the fermentation broth was controlled at 0.5–1%. The fermentation period was 60 hours. Fermentation medium: 1.6g/L magnesium sulfate heptahydrate, 0.03g/L ferrous sulfate heptahydrate, 0.032g/L manganese sulfate monohydrate, 10g/L ammonium sulfate, 5g/L potassium dihydrogen phosphate, 30g/L glucose , 25g/L corn steep liquor, 2.2g/L betaine, 0.006g/L vitamin B1, 50μg/mL ampicillin, 50μg/mL chloramphenicol.

采用两种氨水流加策略:Two ammonia flow addition strategies were used:

种子接入30L发酵罐进行发酵,当OUR、CER达到140m mol L-1h-1时,启动氨水流加,氨水的流加速度根据实时的OUR和CER,使得产酸期OUR和CER保持在60–80m mol L-1h-1The seeds were fermented in a 30L fermenter. When the OUR and CER reached 140 mmol L -1 h -1 , the ammonia water flow was started. The flow rate of the ammonia water was based on the real-time OUR and CER, so that the OUR and CER in the acid production period were kept at 60. -80 mmol L -1 h -1 ;

种子接入30L发酵罐进行发酵,当OUR、CER达到140m mol L-1h-1时,启动氨水流加,用30%氢氧化钠溶液调控发酵液的pH值,氨水的流加速度根据实时的OUR和CER,使得产酸期OUR和CER保持在120–140m mol L-1h-1,两种方案的发酵过程实时参数如图1和图2所示。发酵进行60h,发酵结果如表1所示。The seeds were fermented in a 30L fermenter. When OUR and CER reached 140 mmol L -1 h -1 , the ammonia flow was started, and the pH value of the fermentation broth was adjusted with 30% sodium hydroxide solution. OUR and CER were kept at 120–140 mmol L -1 h -1 during the acid production period. The real-time parameters of the fermentation process of the two schemes are shown in Figures 1 and 2. The fermentation was carried out for 60h, and the fermentation results are shown in Table 1.

表1不同调控策略对应的戊二酸发酵结果比较Table 1 Comparison of glutaric acid fermentation results corresponding to different regulation strategies

Figure BDA0003608521380000061
Figure BDA0003608521380000061

实施例2Example 2

基于微生物生理参数OUR、CER进行反馈调控硫酸铵溶液流加速度Feedback regulation of ammonium sulfate solution flow acceleration based on microbial physiological parameters OUR and CER

按照实施例1的流程和方法,不同处是采用流加50%的硫酸铵提供戊二酸合成的氮源,用30%氢氧化钠溶液调控发酵液的pH值。氮源流加速度根据实时的OUR、CER进行反馈调节,共三种方案:According to the process and method of Example 1, the difference is that the nitrogen source for glutaric acid synthesis is provided by stream-feeding 50% ammonium sulfate, and the pH value of the fermentation broth is regulated with 30% sodium hydroxide solution. The nitrogen source flow acceleration is feedback adjusted according to the real-time OUR and CER. There are three schemes:

Scheme1:种子接入30L发酵罐进行发酵,当OUR、CER达到140m mol L-1h-1时,启动氨水流加,硫酸铵溶液的流加速度根据实时的OUR和CER,使得产酸前期OUR和CER保持在160–180m mol L-1h-1,产酸中后期OUR和CER保持在60–80m mol L-1h-1Scheme1: The seeds are connected to a 30L fermenter for fermentation. When OUR and CER reach 140 mmol L -1 h -1 , the ammonia water flow is started, and the flow acceleration of ammonium sulfate solution is based on the real-time OUR and CER, so that OUR and CER in the early stage of acid production are CER was kept at 160–180 mmol L -1 h -1 , and OUR and CER were kept at 60–80 mmol L -1 h -1 in the middle and late stages of acidogenesis.

Scheme2:种子接入30L发酵罐进行发酵,当OUR、CER达到140m mol L-1h-1时,启动氨水流加,硫酸铵溶液的流加速度根据实时的OUR和CER,使得产酸前期OUR和CER保持在140–160m mol L-1h-1,产酸中后期OUR和CER保持在80–100m mol L-1h-1Scheme2: The seeds are connected to a 30L fermenter for fermentation. When OUR and CER reach 140 mmol L -1 h -1 , the ammonia water flow is started, and the flow acceleration of the ammonium sulfate solution is based on the real-time OUR and CER, so that OUR and CER in the early stage of acid production are CER was kept at 140–160 mmol L -1 h -1 , and OUR and CER were kept at 80–100 mmol L -1 h -1 in the middle and late stages of acidogenesis.

Scheme3:种子接入30L发酵罐进行发酵,当OUR、CER达到140m mol L-1h-1时,启动氨水流加,硫酸铵溶液的流加速度根据实时的OUR和CER,使得产酸期OUR和CER保持在160–180m mol L-1h-1Scheme3: The seeds are connected to a 30L fermenter for fermentation. When OUR and CER reach 140 mmol L -1 h -1 , the ammonia water flow is started, and the flow acceleration of the ammonium sulfate solution is based on the real-time OUR and CER, so that the acid production period OUR and The CER was maintained at 160–180 mmol L -1 h -1 .

三种方案的发酵过程实时参数如图4、5、7所示。如图4、5、6说明,当采用高的OUR、CER来反馈调控氮源流加速度时,戊二酸产酸速率更快。按照Scheme3进行氮源流加速度调控,发酵进行60h,发酵液中戊二酸产率和糖酸转化率最高,分别为53.65g/L和46.76%。The real-time parameters of the fermentation process of the three schemes are shown in Figures 4, 5, and 7. As shown in Figures 4, 5, and 6, when high OUR and CER are used to feedback control the nitrogen source flow acceleration, the acid production rate of glutaric acid is faster. According to Scheme3, the nitrogen source flow rate was adjusted and the fermentation was carried out for 60h. The glutaric acid yield and sugar-acid conversion rate in the fermentation broth were the highest, which were 53.65g/L and 46.76%, respectively.

Claims (10)

1.一种基于微生物生理参数进行反馈调控氮源流加的戊二酸生产方法,包括:1. a method for producing glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition, comprising: 将菌种接种于含有发酵培养基的发酵罐内,以实时的微生物生理参数为依据反馈调控氮源流加,进行发酵获得戊二酸;The strain is inoculated in a fermenter containing a fermentation medium, and the nitrogen source is fed back and controlled based on real-time microbial physiological parameters, and glutaric acid is obtained by fermentation; 其中,微生物生理参数包括氧气消耗速率OUR、二氧化碳生成速率CER。Among them, the microbial physiological parameters include the oxygen consumption rate OUR and the carbon dioxide production rate CER. 2.根据权利要求1所述方法,其特征在于,随着发酵的进行,OUR、CER上升到140–180mmol L-1h-1,在该时,启动氮源流加。2 . The method according to claim 1 , wherein as the fermentation progresses, OUR and CER rise to 140-180 mmol L -1 h -1 , and at this time, the nitrogen source feed is started. 3 . 3.根据权利要求1所述方法,其特征在于,所述调控氮源流加为调控氮源流加的速度,氮源流加速度是基于实时的OUR、CER自动反馈调节,或根据实时的OUR、CER手动反馈调节。3. method according to claim 1, is characterized in that, described regulation and control nitrogen source flow is added as the speed of regulation nitrogen source flow, and nitrogen source flow acceleration is based on real-time OUR, CER automatic feedback regulation, or according to real-time OUR, CER manually feedback regulation. 4.根据权利要求3所述方法,其特征在于,当发酵过程中的OUR、CER低于140–180m molL-1h-1,提高氮源流加速度,使得OUR、CER保持在140-180m mol L-1h-14. The method according to claim 3, characterized in that, when OUR and CER in the fermentation process are lower than 140-180 mmol L -1 h -1 , the nitrogen source flow acceleration is increased, so that OUR and CER are kept at 140-180 mmol L -1 h -1 . 5.根据权利要求1所述方法,其特征在于,所述氮源为氨水、液氨、硫酸铵溶液、氯化铵溶液、尿素溶液、碳酸铵、碳酸氢铵溶液中的一种或几种。5. method according to claim 1, is characterized in that, described nitrogen source is one or more in ammoniacal liquor, liquid ammonia, ammonium sulfate solution, ammonium chloride solution, urea solution, ammonium carbonate, ammonium bicarbonate solution . 6.根据权利要求1所述方法,其特征在于,发酵过程中还包括流加葡萄糖溶液,使得发酵液中葡萄糖的浓度为0.5–1%(w/v)。6 . The method according to claim 1 , wherein the fermentation process further comprises feeding a glucose solution so that the concentration of glucose in the fermentation broth is 0.5-1% (w/v). 7 . 7.根据权利要求6所述方法,其特征在于,所述流加葡萄糖溶液,其中葡萄糖溶液的浓度为50–70%(w/v)。7 . The method according to claim 6 , wherein the glucose solution is fed in, wherein the concentration of the glucose solution is 50-70% (w/v). 8 . 8.根据权利要求1所述方法,所述发酵工艺参数包括:接种量10–20%,发酵温度35–37℃,控制发酵液pH值为6.7–7.2,搅拌转速和溶氧DO关联,使得DO维持在20–40%,罐压保持在0.05–0.1MPa,发酵周期为50–70h。8 . The method according to claim 1 , wherein the fermentation process parameters comprise: the inoculum size is 10-20%, the fermentation temperature is 35-37° C., the pH value of the fermentation broth is controlled to be 6.7-7.2, and the stirring speed is related to dissolved oxygen DO so that the DO was maintained at 20–40%, tank pressure was maintained at 0.05–0.1 MPa, and the fermentation period was 50–70 h. 9.根据权利要求1所述方法,其特征在于,所述发酵培养基包括:3–8g/L磷酸二氢钾、1–3g/L硫酸镁、0.01–0.03g/L硫酸亚铁、0.01–0.04g/L硫酸锰、5–15g/L硫酸铵、20–40g/L葡萄糖、5–30g/L玉米浆,0.5–3g/L甜菜碱,0.002–0.006g/L维生素B1、培养基中添加抗生素,其中所述抗生素为30–200μg/mL氨苄青霉素、30–100μg/mL氯霉素、30–200μg/mL卡那霉素的一种或者几种。The method according to claim 1, wherein the fermentation medium comprises: 3-8g/L potassium dihydrogen phosphate, 1-3g/L magnesium sulfate, 0.01-0.03g/L ferrous sulfate, 0.01 g/L ferrous sulfate –0.04g/L manganese sulfate, 5–15g/L ammonium sulfate, 20–40g/L glucose, 5–30g/L corn steep liquor, 0.5–3g/L betaine, 0.002–0.006g/L vitamin B1, medium Antibiotics are added to the antibiotics, wherein the antibiotics are one or more of 30-200 μg/mL ampicillin, 30-100 μg/mL chloramphenicol, and 30-200 μg/mL kanamycin. 10.根据权利要求1所述方法,所述菌种为代谢工程改造的大肠杆菌或谷氨酸棒杆菌。10. The method according to claim 1, wherein the bacterial species is metabolically engineered Escherichia coli or Corynebacterium glutamicum.
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