A method for improving the fermentation yield of a carbamoyl tobramycin production strain

Abstract

The application provides a method for improving the fermentation yield of a carbamoyl tobramycin production strain. The method improves the fermentation medium of the carbamoyl tobramycin production strain, and the improved fermentation medium is added with a glucose isomerization product or uses the glucose isomerization product to partially replace the glucose in the original fermentation medium. After the method is used, the shake flask fermentation yield of the same strain is increased from 3.5 g / L to 3.91 g / L, and the yield is increased by 11.7%. The application improves the fermentation yield of carbamoyl tobramycin on the basis of fine-tuning the fermentation medium formula, and does not increase the additional fermentation cost, so the application has important industrial application prospects.

Description

TECHNICAL FIELD

[0001] The application relates to a method for improving the fermentation yield of carbamoyl tobramycin, and belongs to the technical field of fermentation engineering. BACKGROUND

[0002] Tobramycin belongs to aminoglycoside antibiotics, which can interfere with the translation process of bacterial ribosomes, affect the synthesis of peptide chains, and produce abnormal proteins to 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. After alkaline hydrolysis and separation and purification treatment of the fermentation liquor, the raw material drug tobramycin can be obtained (J Antibiot 1973, 26, 12, 745-751).

[0003] The growth and reproduction ability of microorganisms, the synthesis efficiency of metabolites, and the yield and quality of products are all affected by the fermentation culture conditions (Biochem Eng J, 2011, 53(2): 187-195). In different stages of fermentation, the growth state of the strain is significantly different, so it is necessary to optimize the culture medium and culture conditions for the strain 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. The important core structure 2-deoxy streptamine of tobramycin molecule is directly derived from glucose, and part of the glucose will participate in the glycosylation modification process of the product in the form of NDP-amino sugar. Part of the glucose added in the fermentation medium component can be directly absorbed and utilized by the bacteria. The macromolecular polysaccharides such as starch and cellulose contained in the medium component need to be gradually hydrolyzed into oligosaccharides by 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] The bacteria can establish a related enzyme system to hydrolyze and utilize the macromolecular polysaccharides to produce glucose in the middle and late stages of fermentation, but it is unknown whether the hydrolysis rate of this complex enzyme system can meet the secondary metabolic demand. The purpose of the present application is to optimize the addition of small molecule carbon sources in the medium component through fermentation engineering, to avoid the metabolic inhibition caused by excessive glucose production, to provide more sufficient substrates for secondary metabolites, to improve the fermentation production level of the strain of carbamoyl tobramycin, and to be used for the industrial production of tobramycin.

[0006] In the early stage of their work, the applicants of the present invention constructed an engineered strain S. tenebrarius Tb-△aprJ-△tobR / ermE*p-tobO that produces a high yield of carbamylated tobramycin (Lin Jianping. A method for increasing the yield of carbamylated tobramycin and a production strain: CN117384922[P]. 2024-01-12.). The authors found that replacing the single glucose in the original fermentation medium with a mixed sugar of glucose and fructose can effectively increase the production level of carbamylated tobramycin.

[0007] In order to further improve the fermentation level of carbamoyl tobramycin, an industrialized method is needed to increase the fermentation yield of carbamoyl tobramycin and reduce the production cost by fermentation optimization of the carbon source of the culture medium. Summary of the Invention

[0008] 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-producing strain. The method comprises: fermenting the carbamycin-producing strain to produce carbamycin in an improved fermentation medium, wherein a glucose isomerization product is added to the improved fermentation medium, 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 products 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; and 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; and 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 carbamylated tobramycin producing strain. Preferably, the carbamylated tobramycin producing strain is a carbamylated tobramycin high-yielding strain. More preferably, the carbamylated tobramycin high-yielding strain is constructed by inactivating the tobR gene in a strain S. tenebrarius in which the aprJ gene is knocked out, and overexpressing the gene encoding 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 includes the following components: soybean powder, corn flour, glucose, silkworm pupa powder, fish meal, calcium chloride, calcium carbonate, soybean oil, and valine.

[0015] Furthermore, the improved fermentation medium includes the following components in percentage by mass: 5% soybean powder, 2% corn flour, 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 carbamyl tobramycin had an ability to utilize fructose and allulose, simple isomerization products of glucose, although not as good as glucose, but significantly better than macromolecular polysaccharides such as starch and cellulose; when a mixture of fructose and glucose was used instead of single glucose, the fermentation yield of carbamyl tobramycin was 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 that of 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 The figure shows the measurement results of various fermentation parameters during shake flask fermentation;

[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 the result of the shake flask fermentation experiment with the addition of small molecule sugars;

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

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

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

[0027] The carbamoyl tobramycin-producing strain used in the following examples is S. tenebrarius Tb-△aprJ-△tobR / ermE*p-tobO. The construction method of this strain is described in CN117384922A. It is obtained by inactivating the tobR gene in the aprJ gene-knockout strain S. tenebrarius and overexpressing the gene encoding 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. It should be noted that the method of the present invention is applicable to any carbamoyl tobramycin-producing strain.

[0028] The above-mentioned strains were cultured upside down on solid MS plates (2% soybean flour, 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 flour, 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 flour, 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 cultured at 37°C, 220 rpm, with shaking for 24 h. The culture medium was then transferred to fermentation medium and cultured at 37°C, 220 rpm, with shaking for 144 h. After fermentation, the supernatant obtained by centrifugation was diluted to a predetermined ratio and derivatized with 2,4-dinitrofluorobenzene. The reaction solution was filtered through a 0.22 μm organic filter and analyzed by high-performance liquid chromatography (HPLC) using a C-18 column (Hypersil BDS 5 μm, 4.6 mm × 250 mm) with detection at 365 nm. The mobile phase was a 0.01 mM aqueous ammonium acetate solution (pH adjusted to 4.0 with phosphoric acid) / acetonitrile (47:53, v / v) at a flow rate of 1 mL / min. All results were averaged using three replicates.

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

[0033] During the shake flask fermentation culture process, samples were taken every 24 hours to measure 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 a suitable 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 in the figure, during the fermentation process, the accumulation curve of the product carbamoyl tobramycin is shown in the red curve, the system glucose content is shown in the black curve, and the biomass accumulation is shown in the blue curve. It was observed that during the first 48 hours of fermentation, glucose was consumed in large quantities, and the bacterial biomass accumulated rapidly. After 48 hours, the system glucose concentration remained at a low level of 0.2-0.5 g / L. At this time, biomass accumulation slowed, and the system gradually transitioned from primary metabolism to secondary metabolism, accompanied by the rapid accumulation of the product carbamoyl tobramycin.

[0034] Example 3: Supplementation of glucose during 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 in 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 in the data, after glucose was supplemented for 48 hours in each experimental group, 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, as excessive glucose may 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 psicose 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, when no glucose was added to the culture medium, the fermentation yield of carbamoyl tobramycin was only 2.29 g / L, only 65.46% of the 3.5 g / L in the control group. The glucose required for metabolism was entirely 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 groups where fructose and psicose 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 addition. It is believed that the bacteria's utilization of glucose isomerization products was relatively slow, and the fermentation performance was not as good as that of glucose. It is speculated that this may be because the establishment of relevant isomerases in the bacteria requires a certain amount of time, and the utilization of glucose isomerization products is better than that of macromolecular carbohydrates, resulting in better fermentation results than the experimental group without monosaccharide addition.

[0039] Example 5: Observation of the growth status of shake flask fermentation bacteria:

[0040] 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 fructose added as an example, it was observed that in all experimental groups without glucose addition, the mycelium grew more slowly in the early stage of fermentation, and fewer mycelium adhered to the bottle wall, suggesting that glucose is 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 mixture 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 6.75g / L glucose group was slightly lower than that in the control group. However, increasing the glucose concentration to 8.25g / L was sufficient for bacterial metabolism and achieved the same effect as 15g / L glucose. Furthermore, as the concentration of the mixed sugars increased, the fermentation yield showed an initial increase followed by a decrease. The yields of the experimental groups supplemented with mixed sugars No. 1 were generally higher than those of mixed sugars No. 2. The group supplemented with 30g / L of mixed sugars No. 1 exhibited the highest tobramycin yield, reaching 3.91g / L, an 11.6% increase compared to 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 scope of protection 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 carbamycin-producing strain to produce carbamycin in an improved fermentation medium, wherein the carbamycin-producing strain is Streptomyces tenebrarius; The improved fermentation medium is supplemented 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; In the improved fermentation medium, the glucose isomerization product is fructose, the mass ratio of fructose to glucose is 9:11, and the total concentration of fructose and glucose is 15-30 g / L; Alternatively, in the improved fermentation medium, the glucose isomerization products are fructose and psicose, and the mass percentages of glucose, fructose, and psicose in the mixed sugars in the fermentation medium are: 45.4% glucose, 37.2% fructose, and 17.4% psicose, and the total concentration of glucose isomerization products and glucose is 15 to 22.5 g / L.

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

3. The method according to claim 2, characterized in that The carbamyl tobramycin high-yield strain is constructed by the following method: inactivating the tobR gene in the aprJ gene-knocked strain S. tenebrarius, and overexpressing the coding gene of the TauD / TfdA family oxidase.

4. The method according to claim 1, wherein 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.

5. The method according to claim 1, wherein The improved fermentation medium comprises the following components in percentage by mass: 5% soybean powder, 2% corn flour, 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.

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

Images