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

Abstract

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.

Description

A method for the production of glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition

technical field

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.

Background technique

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.

SUMMARY OF THE INVENTION

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.

A method for producing glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition, comprising:

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).

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.

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.

Further, 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 .

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.

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).

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.

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).

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.

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.

The strain is metabolically engineered Escherichia coli or Corynebacterium glutamicum.

Note: The mass volume percent (w/v) in the present invention is all g/100ml.

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).

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.

Bioprocess glutaric acid fermentation includes glycerol tube seed activation, shake flask seed culture, seed tank seed culture and fermentation process.

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.

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.

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.

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.

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.

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. .

beneficial effect

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.

Description of drawings

Figure 1 shows the deamination and decarboxylation reactions in the synthesis of glutaric acid based on the AMV pathway.

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.

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.

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 .

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 .

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.

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.

Detailed ways

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.

Example 1

Feedback regulation of ammonia flow acceleration based on microbial physiological parameters OUR and CER

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) 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) 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) 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) 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.

Two ammonia flow addition strategies were used:

The 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 ;

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.

Table 1 Comparison of glutaric acid fermentation results corresponding to different regulation strategies

Example 2

Feedback regulation of ammonium sulfate solution flow acceleration based on microbial physiological parameters OUR and CER

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: 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: 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: 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 .

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. a method for producing glutaric acid based on microbial physiological parameters for feedback regulation of nitrogen source stream addition, comprising: 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; Among them, the microbial physiological parameters include the oxygen consumption rate OUR and the carbon dioxide production rate CER. 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. 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. 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. 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 . 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 . 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 . 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. 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. The method according to claim 1, wherein the bacterial species is metabolically engineered Escherichia coli or Corynebacterium glutamicum.

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