Method for improving fermentation yield of carbamoyl tobramycin production strain

Abstract

The invention provides a method for improving the fermentation yield of a carbamoyl tobramycin production strain. According to the method, a culture medium in the fermentation process of a carbamoyl tobramycin producing strain is improved, a glucose isomerization product is added into the improved fermentation culture medium, or the glucose isomerization product is used for partially replacing glucose in an original fermentation culture medium. After the method is used, the shake flask fermentation yield of carbamoyl tobramycin of the same strain is increased to 3.91 g / L from 3.5 g / L, and is increased by 11.7%. On the basis of finely adjusting the formula of the fermentation culture medium, the fermentation yield of carbamoyl tobramycin is increased while the additional fermentation cost is basically not increased, and the method has important industrial application prospects.

Description

Technical Field

[0001] The invention relates to a method for improving the fermentation yield of carbamyl tobramycin, and belongs to the technical field of fermentation engineering. Background Art

[0002] Tobramycin belongs to the aminoglycoside antibiotics, which can interfere with the translation process of bacterial ribosomes, affect peptide chain synthesis, and produce abnormal proteins that kill bacteria (Nature Communications 2015, 6). In the industrial production process, Streptoalloteichus tenebrarius can ferment to produce a series of aminoglycoside secondary metabolites such as carbamoyl tobramycin, and the fermentation broth can be treated with alkaline hydrolysis and separation and purification to obtain the raw material tobramycin (J Antibiot 1973, 26, 12, 745-751).

[0003] The growth and reproduction ability of microorganisms, the efficiency of metabolite synthesis, and the yield and quality of products are all affected by fermentation culture conditions (Biochem Eng J, 2011, 53(2): 187-195). At different stages of fermentation, the growth state of strains varies significantly, so it is necessary to optimize the culture medium and culture conditions for strains in different growth states (Process Biochem, 2004, 39(9): 1057-1062).

[0004] Among them, glucose is an important substrate for the biosynthesis of aminoglycoside compounds. 2-deoxystreptamine, an important core structure of tobramycin molecules, is directly derived from glucose. In addition, part of the glucose will participate in the glycosylation modification process of the product in the form of NDP-amino sugars. Some of the glucose added to the fermentation medium components can be directly absorbed and utilized by the bacteria. The macromolecular polysaccharides such as starch and cellulose contained in the culture medium components need to be gradually hydrolyzed into oligosaccharides with the participation of a series of amylases or cellulases, and finally absorbed and utilized by the bacteria in the form of glucose (Braz J Microbiol, 2010, 41(4): 850-861; J Nat Prod, 2020, 83(6): 2054-2055).

[0005] In the middle and late stages of fermentation, the bacteria can establish a related enzyme system to hydrolyze macromolecular polysaccharides to produce glucose, but it is still unknown whether the hydrolysis rate of this complex enzyme system can meet the needs of secondary metabolism. The purpose of the present invention is to optimize the addition of small molecular carbon sources in the culture medium components through fermentation engineering, while avoiding metabolic inhibition caused by excessive glucose, provide more sufficient substrates for secondary metabolites, and improve the fermentation production level of carbamoyl tobramycin of the strain for industrial production of tobramycin.

[0006] In the previous work, the applicant of the present invention constructed an engineered strain S. tenebrarius Tb-△aprJ-△tobR / ermE*p-tobO with high yield of carbamoyl tobramycin (Lin Jianping. A method for increasing the yield of carbamoyl tobramycin and a production strain: CN117384922[P]. 2024-01-12.). After research, it was found that the use of a mixed sugar of glucose-fructose to replace the single glucose in the original fermentation medium components can effectively improve the production level of carbamoyl tobramycin.

[0007] In order to further improve the fermentation level of carbamycin tobramycin, an industrial method is needed to improve the fermentation yield of carbamycin tobramycin and reduce the production cost through fermentation optimization of the carbon source of the culture medium. Summary of the invention

[0008] In order to solve the problems in the prior art, the present invention proposes a method for increasing the yield of carbamyl tobramycin. The present invention adopts the following technical solutions to solve the above problems:

[0009] The present invention provides a method for improving the fermentation yield of a carbamycin tobramycin producing strain, the method comprising: fermenting the carbamycin tobramycin producing strain in an improved fermentation medium to produce carbamycin tobramycin, wherein the improved fermentation medium is added with a glucose isomerization product, or the improved fermentation medium is obtained by partially replacing the glucose in the original fermentation medium with the glucose isomerization product.

[0010] According to an embodiment of the present invention, in the improved fermentation medium, the concentration of glucose is 5 to 37.5 g / L; the concentration of glucose isomerization product is 5 to 37.5 g / L.

[0011] According to an embodiment of the present invention, the glucose isomerization product is one or more of fructose and psicose; in the improved fermentation medium, the mass ratio of the glucose isomerization product to glucose is 3:7 to 7:3.

[0012] Furthermore, the glucose isomerization product is fructose; in the improved fermentation medium, the mass ratio of fructose to glucose is 9:11.

[0013] The method of the present invention is applicable to any carbamoyl tobramycin producing strain. Preferably, the carbamoyl tobramycin producing strain is a carbamoyl tobramycin high-producing strain. More preferably, the carbamoyl tobramycin high-producing strain is constructed by the following method: inactivating the tobR gene in the strain S. tenebrarius in which the aprJ gene is knocked out, and overexpressing the coding gene of the TauD / TfdA family oxidase. The nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2.

[0014] According to an embodiment of the present invention, the improved fermentation medium comprises the following components: soybean powder, corn powder, glucose, silkworm pupa powder, fish meal, calcium chloride, calcium carbonate, soybean oil and valine.

[0015] Furthermore, the improved fermentation medium comprises the following components in percentage by mass: 5% soybean powder, 2% corn powder, 0.825% glucose, 0.675% glucose, 0.6% silkworm pupa powder, 1% fish meal, 0.82% ammonium chloride, 0.025% calcium carbonate, 3% soybean oil and 0.117% valine.

[0016] The advantages of the present invention compared to the prior art are:

[0017] The present invention found that the industrial production strain of carbamoyl tobramycin has a lower utilization capacity for fructose and allulose, simple isomerization products of glucose, than glucose, but is significantly better than macromolecular polysaccharides such as starch and cellulose; when a mixed sugar of fructose and glucose is used instead of single glucose, the fermentation yield of carbamoyl tobramycin is improved.

[0018] Among them, when no glucose was added to the culture system at all, the fermentation yield of carbamycin tobramycin decreased by 34.6%; when an equal amount of 15 g / L fructose was used instead of glucose, the fermentation yield of carbamycin tobramycin decreased by 22.6%; when an equal amount of 15 g / L psicose was used instead of glucose, the fermentation yield of carbamycin tobramycin decreased by 23.4%; this shows that although the utilization of fructose and psicose by this carbamycin production strain is not as good as glucose, it is significantly better than the blank control group without glucose addition.

[0019] Among them, when 30 g / L of a mixed sugar composed of 55% glucose and 45% fructose was used to replace the original 15 g / L glucose in the culture system, the yield of carbamyl tobramycin was the highest, increasing by 11.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the measurement result diagram of each fermentation parameter in the shake flask fermentation process;

[0021] Figure 2 Schematic diagram of the effect of glucose addition on fermentation after 48h of fermentation;

[0022] Figure 3 Schematic diagram of the isomerization reaction of D-glucose, D-fructose and D-psicose;

[0023] Figure 4 This is a graph showing the results of a shake flask fermentation experiment with the addition of small molecule sugars;

[0024] Figure 5 Schematic diagram of the growth of mycelium when different sugars are added;

[0025] Figure 6 This is a graph showing the optimization results for mixed sugar concentration in shake flask fermentation. DETAILED DESCRIPTION

[0026] The present invention is described below in conjunction with a specific embodiment. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited thereto:

[0027] The carbamoyl tobramycin production strain used in the following examples is S. tenebrarius Tb-△aprJ-△tobR / ermE*p-tobO. The construction method of the strain is described in CN117384922A, which is obtained by inactivating the tobR gene in the aprJ gene-knocked strain S. tenebrarius and overexpressing the coding gene of the TauD / TfdA family oxidase, wherein the nucleotide sequence of the tobR gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2. It should be noted that the method of the present invention is applicable to any carbamoyl tobramycin production strain.

[0028] The above-mentioned strains were cultured upside down on solid MS plates (2% soybean powder, 2% mannose and 2% agar powder) in a constant temperature incubator at 37°C, and cultured in YEME liquid medium (0.3% yeast powder, 0.3% malt extract, 2.5% sucrose, 0.5% peptone and 1% glucose), shake flask fermentation seed medium (2% soybean powder, 0.3% casein, 0.5% glucose, 1% dextrin, 0.1% potassium chloride, 0.025% calcium chloride, 0.025% potassium dihydrogen phosphate and 0.5% magnesium sulfate) and shake flask fermentation medium (5% soybean powder, 2% corn flour, 1.5% glucose, 0.6% silkworm pupa powder, 1% fish meal, 0.82% ammonium chloride, 0.025% light calcium carbonate, 3% soybean oil and 0.117% valine).

[0029] The biological reagents used in the following examples were purchased from the reagent company

[0030] Example 1: HPLC detection of fermentation products

[0031] The activated strain was inoculated into a shake flask fermentation seed medium, and after shaking culture at 37°C and 220rpm for 24h, the fermentation medium was transferred to 37°C and 220rpm for 144h. After the fermentation, the supernatant obtained by centrifugation was diluted a certain multiple, and derivatized with 2,4-dinitrofluorobenzene. The reaction solution was filtered with a 0.22μm organic filter membrane and analyzed by high performance liquid chromatography (HPLC). A C-18 column (Hypersil BDS 5μm, 4.6mm×250mm) was used, and the detection was performed at a wavelength of 365nm. A 0.01mM ammonium acetate aqueous solution (pH adjusted to 4.0 with phosphoric acid) / acetonitrile (47:53, v / v) was used as the mobile phase, and the flow rate was 1mL / min. All results were repeated three times and the average value was taken.

[0032] Example 2: Determination of parameters during shake flask fermentation

[0033] During the shake flask fermentation culture, samples were taken every 24 hours to determine the product accumulation of carbamoyl tobramycin, the consumption of glucose, and the accumulation of biomass during the fermentation process. The supernatant obtained by centrifugation was diluted a certain multiple, and the yield of the product carbamoyl tobramycin was determined by HPLC. The supernatant was diluted an appropriate multiple, and the content of glucose in the system was determined by glucose enzyme membrane. The precipitate obtained by centrifugation was weighed and subtracted from the control group at 0 hours of fermentation. The difference was roughly recorded as the accumulation of biomass during the fermentation process. All results were repeated three times and the average value was taken. Figure 1 As shown, during the fermentation process, the accumulation curve of the product carbamoyl tobramycin is shown as the red curve, the content of glucose in the system is shown as the black curve, and the accumulation of biomass is shown as the blue curve. It was observed that in the first 48 hours of fermentation, glucose was consumed in large quantities and the bacterial biomass accumulated rapidly. After 48 hours, the glucose concentration in the system was maintained at a low level of 0.2-0.5 g / L. At this time, the biomass accumulated slowly, gradually transforming from primary metabolism to secondary metabolism, accompanied by the rapid accumulation of the product carbamoyl tobramycin.

[0034] Example 3: Supplementation of glucose in shake flask fermentation for 48 h

[0035] Different concentrations of glucose were added during shake flask fermentation for 48 h, and the final glucose concentrations of the fermentation system were set to 15 g / L, 30 g / L, 45 g / L and 60 g / L, respectively. An equal amount of sterile water was added to the control group. Figure 2As shown, after glucose was supplemented in each experimental group for 48 hours, the biosynthesis of the product carbamoyl tobramycin was inhibited, and the higher the glucose concentration, the more significant the inhibitory effect. Within the measurement range, the final fermentation yield of the experimental group with 60 g / L glucose was only 24% of that of the control group. After the fermentation entered the secondary metabolism stage, it was not suitable to add a large amount of glucose, and too much glucose might interfere with the secondary metabolic process.

[0036] Example 4: Addition of different small molecule sugars to the culture medium

[0037] Try to replace glucose with glucose isomers in the fermentation system. Figure 3 As shown, fructose is a one-step isomerization product of glucose, and allulose is a two-step isomerization product of glucose. Both are soluble small molecule carbon sources, and their solubility will not become a limiting factor for fermentation utilization.

[0038] Fructose, allulose and blank experiments were used to replace glucose components. Figure 4 As shown in the figure, when glucose was not added to the culture medium, the fermentation yield of carbamoyl tobramycin was only 2.29 g / L, which was only 65.46% of the 3.5 g / L in the control group. The glucose required for metabolism was completely derived from the hydrolysis of macromolecular carbohydrates. It is speculated that the hydrolysis rate was insufficient to supply bacterial growth and product synthesis, limiting the synthesis of secondary metabolites. In the experimental group where fructose and allulose replaced glucose, it was found that the yield of carbamoyl tobramycin was between the control group with 15 g / L glucose and the experimental group without any monosaccharide added. It is believed that the bacteria's utilization of glucose isomerization products is relatively slow, and the fermentation is not as good as glucose. It is speculated that it may be because the establishment of related isomerases in the bacteria takes a certain amount of time, and the utilization of glucose isomerization products is better than macromolecular carbohydrates, and the fermentation results are better than those of the experimental group without adding monosaccharides.

[0039] Example 5: Observation of the growth status of the fermentation cells in shake flasks:

[0040] In order to determine the effects of glucose isomers and glucose on the growth of bacteria during shake flask fermentation, the growth of bacteria in shake flasks containing 15 g / L fructose and 15 g / L glucose was compared. Figure 5 Taking the experimental group with added fructose as an example, it was observed that in all experimental groups without added glucose, the mycelium grew slowly in the early stage of fermentation, and there were fewer mycelium attached to the bottle wall, which indicated that glucose was indispensable in the early stage of fermentation.

[0041] Example 6: Addition of small molecule mixed sugars to the culture medium

[0042] Two mixed sugars were used to replace the 15g / L glucose in the culture medium. Mixed sugar No. 1 was a mixed system of 55% glucose and 45% fructose, and mixed sugar No. 2 was 45.4% glucose, 37.2% fructose and 17.4% allulose. 15g / L glucose was set as the control group, and 8.25g / L and 6.75g / L glucose were used as positive controls for the addition of 15g / L mixed sugar No. 1 and No. 2. Figure 6 As shown, the fermentation yield of carbamoyl tobramycin in the group with 6.75g / L glucose was slightly lower than that in the control group, while when the glucose concentration increased to 8.25g / L, it could basically meet the metabolism of the bacteria and achieve the same effect as 15g / L glucose. In addition, as the concentration of mixed sugar added gradually increased, the fermentation yield showed a trend of first increasing and then decreasing. The yield of the experimental group with the addition of mixed sugar No. 1 was generally higher than that of mixed sugar No. 2, among which the yield of carbamoyl tobramycin in the experimental group with the addition of 30g / L mixed sugar No. 1 was the highest, reaching 3.91g / L, an increase of 11.6% over the initial culture medium.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing the fermentation yield of a tobramycin carbamyl producing strain, characterized in that: The method comprises: fermenting a carbamoyl tobramycin-producing strain in an improved fermentation medium to produce carbamoyl tobramycin, wherein the improved fermentation medium is added with a glucose isomerization product, or the improved fermentation medium is obtained by partially replacing the glucose in the original fermentation medium with the glucose isomerization product.

2. The method according to claim 1, characterized in that In the improved fermentation medium, the concentration of glucose is 5 to 37.5 g / L; the concentration of glucose isomerization product is 5 to 37.5 g / L.

3. The method according to claim 1, characterized in that The glucose isomerization product is one or more of fructose and psicose; in the improved fermentation medium, the mass ratio of the glucose isomerization product to glucose is 3:7 to 7:

3.

4. The method according to claim 1, characterized in that The glucose isomerization product is fructose; in the improved fermentation medium, the mass ratio of fructose to glucose is 9:

11.

5. The method according to claim 1, characterized in that: The carbamycin-producing strain is a carbamycin-producing strain with high yield.

6. The method according to claim 5, characterized in that The carbamyl tobramycin high-producing strain is constructed by the following method: inactivating the tobR gene in the aprJ gene knocked out strain S. tenebrarius, and overexpressing the coding gene of the TauD / TfdA family oxidase.

7. The method according to claim 1, characterized in that The improved fermentation medium comprises the following components: soybean powder, corn powder, glucose, silkworm pupa powder, fish meal, calcium chloride, calcium carbonate, soybean oil and valine.

8. The method according to claim 1, characterized in that The improved fermentation medium comprises the following components in percentage by mass: 5% soybean powder, 2% corn powder, 0.825% glucose, 0.675% fructose, 0.6% silkworm pupa powder, 1% fish meal, 0.82% ammonium chloride, 0.025% calcium carbonate, 3% soybean oil and 0.117% valine.

9. The method according to claim 1, characterized in that: The fermentation production conditions are: 37°C, 220rpm shake flask fermentation.

Images