A spiral mycin fermentation method based on nitrogen source regulation

Abstract

The application discloses a spiramycin fermentation method based on nitrogen source regulation, and relates to the technical field of spiramycin fermentation. The application improves the spiramycin fermentation titer and reduces the generation levels of impurities D and F without changing the production strain, introducing complex genetic modification and large equipment modification, by adjusting the ratio of the available inorganic nitrogen source and the composite organic nitrogen source in the initial culture medium, and optimizing the composition of the organic nitrogen source in the feeding stage. The application can solve the problems in the existing mature fermentation process, such as the limited titer improvement space, the possible influence of the slow-release organic nitrogen source such as soybean meal on impurity F in the feeding stage, the mismatch between the nitrogen metabolism rhythm and the secondary metabolism start caused by the too fast use of the available ammonium salt, and the high impurity D level in the late fermentation stage. The application also verifies the action mechanism of the nitrogen source regulation process by the changes of the total sugar, amino nitrogen and related biosynthesis gene expression in the fermentation process, and provides a basis for subsequent process amplification and quality control.

Description

Technical Field

[0001] This invention relates to the field of spiramycin fermentation technology, specifically a nitrogen-source-controlled fermentation method for spiramycin. Background Technology

[0002] Spiramycin is a 16-membered macrolide antibiotic produced by fermentation of actinomycetes such as Streptomyces disaccharides. Its molecular structure consists of a macrolide backbone and multiple deoxyglycosylation modifications. During fermentation, cell growth, precursor supply, glycosylation synthesis, and glycosylation transfer collectively determine the level of spiramycin synthesis and the composition of related impurities.

[0003] In existing industrial or pilot-scale fermentation processes, the culture medium typically contains carbohydrate carbon sources, inorganic salts, inorganic ammonium salts, and organic nitrogen sources such as corn steep liquor and soybean meal. Inorganic ammonium salts are relatively readily available nitrogen sources and are easily utilized in the early stages of fermentation. Complex organic nitrogen sources such as corn steep liquor and soybean meal provide nitrogen, amino acids, vitamins, and trace growth factors, but the release rate, amino acid composition, and metabolic effects of different organic nitrogen sources vary.

[0004] Simply adjusting inorganic salt components such as NaCl / KCl, phosphates, and zinc salts has limited effect on improving potency, and under certain conditions may even increase the risk of impurity D formation. In contrast, the ammonium salt level and the composition of nitrogen sources such as corn steep liquor and soybean meal have a more significant impact on potency and impurities D and F. Therefore, optimizing the fermentation process around the nitrogen source structure is more suitable as a key entry point for improving spiramycin potency and controlling impurities.

[0005] In existing processes, while adding soybean meal during the feeding stage can maintain a certain supply of slow-release nitrogen, its insoluble components and protein degradation process may alter the nitrogen metabolism state in the later stages of fermentation. Furthermore, if the initial ammonium salt level is too high, the consumption of readily available nitrogen by the cells in the early stages will prolong the primary metabolic dominance phase, delaying the utilization of organic nitrogen and the initiation of secondary metabolism, which is detrimental to the product synthesis rhythm and impurity control. Therefore, this invention provides a nitrogen-source-regulated fermentation method for spiramycin. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nitrogen-source-controlled fermentation method for spiramycin to solve the aforementioned problems.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a nitrogen-source-controlled fermentation method for spiramycin, comprising the following steps:

[0008] A1. Preparation of initial culture medium: Based on the existing spiramycin fermentation culture medium, increase the amount of corn steep liquor to 1.25 to 1.40 times the original amount, and reduce the amount of inorganic ammonium salt to 0.60 to 0.80 times the original amount;

[0009] A2. Preparation of supplemental culture medium: Eliminate or significantly reduce the amount of soybean meal powder added;

[0010] A3. Control the fermentation process: Monitor pH, amino nitrogen, total sugar, cell concentration, spiramycin potency, impurity D and impurity F during the fermentation process, and use pH rise and amino nitrogen change as process judgment indicators of nitrogen source utilization rhythm and secondary metabolism initiation state.

[0011] A4. Control fermentation time: The fermentation cycle is 120 hours. After fermentation, the spiramycin potency and impurity composition are tested.

[0012] Preferably, the amount of corn steep liquor used in A1 is 1.40 times that of the original process.

[0013] Preferably, the amount of inorganic ammonium salt used in A1 is 0.70 times that of the original process.

[0014] Preferably, soybean meal powder is not added to the supplemental culture medium described in A2.

[0015] Preferably, the inorganic ammonium salt includes ammonium sulfate, ammonium chloride, or a mixture thereof.

[0016] Preferably, the fermentation process uses Streptomyces dioxinus as the production strain.

[0017] Preferably, it also includes: monitoring the expression levels of genes related to spiramycin biosynthesis during fermentation to verify the mechanism by which nitrogen source regulation affects secondary metabolism.

[0018] Preferably, the biosynthesis-related genes include macrocyclic lactone backbone synthesis genes, deoxyglycosylation genes, and glycosyltransferase genes.

[0019] Preferably, the pH rise is initiated 10 to 15 hours in advance.

[0020] Beneficial effects

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention improves the fermentation potency of spiramycin and reduces the generation levels of impurities D and F by adjusting the ratio of readily available inorganic nitrogen sources to complex organic nitrogen sources in the initial culture medium and optimizing the composition of organic nitrogen sources during the feeding stage, without changing the production strain, introducing complex genetic modifications or large-scale equipment modifications.

[0023] This invention can solve problems such as limited potential for potency improvement in existing mature fermentation processes, the potential impact of slow-release organic nitrogen sources such as soybean meal powder on impurity F during the feeding stage, the mismatch between nitrogen metabolism rhythm and secondary metabolism initiation due to excessively rapid utilization of fast-acting ammonium salts, and the high level of impurity D in the later stages of fermentation.

[0024] This invention also verifies the mechanism of nitrogen source regulation by analyzing changes in the expression of total sugar, amino nitrogen, and related biosynthetic genes during fermentation, providing a basis for subsequent process scale-up and quality control. Attached Figure Description

[0025] Figure 1 This is a flowchart of the process of this invention;

[0026] Figure 2 This is a schematic diagram illustrating the effect of supplemental feeding to remove soybean meal powder on the 15L spiramycin fermentation process in this invention;

[0027] Figure 3 This is a schematic diagram illustrating the effect of increasing the initial corn steep liquor by 25% on the fermentation process of 15L spiramycin according to the present invention;

[0028] Figure 4 This is a schematic diagram illustrating the effect of increasing the initial corn steep liquor by 40% on the fermentation process of 15L of spiramycin according to the present invention;

[0029] Figure 5 This is a schematic diagram illustrating the effect of a 30% reduction in the initial ammonium salt content on the 15L spiramycin fermentation process.

[0030] Figure 6 This is a schematic diagram illustrating the effect of the corn steep liquor reduction process of this invention on the fermentation results of spiramycin 15L;

[0031] Figure 7 This is a schematic diagram illustrating the effect of the optimized nitrogen source structure combination of this invention on the expression of genes related to spiramycin glycosyl modification.

[0032] Figure label:

[0033] Figure 2 In the middle: (A) Changes in potency; (B) Changes in the proportion of impurity F; (C) Changes in the proportion of impurity D; (D) Changes in total sugar; (E) Changes in amino nitrogen; (F) Changes in bacterial concentration; The control tank is based on the original culture medium conditions; The experimental tank is based on the control tank with the soybean meal powder removed from the feed.

[0034] Figure 3 In the experiment: (A) Changes in potency; (B) Changes in the proportion of impurity F; (C) Changes in the proportion of impurity D; (D) Changes in total sugar; (E) Changes in amino nitrogen; (F) Changes in bacterial concentration; The control tank was the basic condition for removing soybean meal powder in the feed; The experimental tank increased the initial corn steep liquor addition by 25% based on the control tank.

[0035] Figure 4 In the experiment: (A) Changes in potency; (B) Changes in the proportion of impurity F; (C) Changes in the proportion of impurity D; (D) Changes in total sugar; (E) Changes in amino nitrogen; (F) Changes in bacterial concentration; The control tank was the basic condition for removing soybean meal powder in the feed; The experimental tank increased the initial corn steep liquor addition by 40% based on the control tank.

[0036] Figure 5 In the experiment: (A) Changes in potency; (B) Changes in the proportion of impurity F; (C) Changes in the proportion of impurity D; (D) Changes in total sugar; (E) Changes in amino nitrogen; (F) Changes in bacterial concentration; The control tank was prepared under the basic conditions of removing soybean meal and increasing the initial corn steep liquor by 40% in the feed; The experimental tank was prepared under the control conditions with the initial ammonium salt addition reduced by 30%;

[0037] Figure 6 In the middle: (A) potency; (B) proportion of impurity F; (C) proportion of impurity D; (D) total sugar; (E) amino nitrogen; (F) bacterial concentration; the control tank is a process of removing soybean meal powder by feeding, and the condition tank further increases corn steep liquor and reduces ammonium salts on this basis. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Please see Figure 1-7 A nitrogen-source-regulated fermentation method for spiramycin includes the following steps:

[0040] A1. Preparation of initial culture medium: Based on the existing spiramycin fermentation culture medium, the amount of corn steep liquor is increased to 1.25 to 1.40 times that of the original process, preferably 1.40 times, and the amount of inorganic ammonium salt is reduced to 0.60 to 0.80 times that of the original process. The inorganic ammonium salt includes ammonium sulfate, ammonium chloride or a mixture thereof, and preferably 0.70 times.

[0041] A2. Preparation of supplemental culture medium: Eliminate or significantly reduce the amount of soybean meal powder added;

[0042] A3. Controlling the fermentation process: Using Streptomyces simulans as the production strain, pH, amino nitrogen, total sugar, cell concentration, spiramycin titer, impurity D, and impurity F were monitored during fermentation. pH rise and amino nitrogen changes were used as indicators to judge the process of nitrogen source utilization rhythm and secondary metabolism initiation status. The pH rise was 10-15 hours in advance. The expression level of spiramycin biosynthesis-related genes was monitored during fermentation to verify the influence mechanism of nitrogen source regulation on secondary metabolism. Among them, biosynthesis-related genes include macrolide skeleton synthesis genes, deoxyglycosylation genes, and glycosyltransferase genes.

[0043] A4. Control fermentation time: The fermentation cycle is 120 hours. After fermentation, the spiramycin potency and impurity composition are tested.

[0044] Example 1: Removal of soybean meal powder during the feeding stage

[0045] In a 15L fermenter, the original fermentation process was used as a control. The inoculation method, fermentation temperature, aeration, stirring, carbon source feeding and other inorganic salt conditions were kept basically the same, except that the soybean cake powder in the feeding medium was removed. The fermentation cycle was adjusted to about 120 hours, and the total sugar, amino nitrogen, cell concentration, pH, potency and impurities D and F were tested regularly.

[0046] The results are as follows Figure 2 As shown, after removing soybean meal during the fed-batch stage, the spiramycin potency increased by approximately 12.9% compared to the control, and the proportion of impurity D decreased by 8.4%. This result indicates that soybean meal removal during the fed-batch stage is not a necessary condition for maintaining high potency; in fact, removing soybean meal is beneficial for improving the quality of the product in the later stages of fermentation, possibly due to a reduction in its insoluble components and a decrease in fluctuations in nitrogen metabolism during the later stages.

[0047] Example 2: Increased initial corn steep liquor dosage

[0048] Based on Example 1, the amount of corn steep liquor in the initial fermentation medium was increased by 25% and 40% respectively (i.e., 1.25 times and 1.40 times the original amount), while other conditions remained unchanged. During the fermentation process, the focus was on observing the pH rise, changes in amino nitrogen, accumulation of potency, and changes in impurities D and F.

[0049] The results are as follows Figure 3 and Figure 4 As shown, when the initial corn steep liquor concentration was increased by 25%, the improvement in potency and impurities was relatively limited. When the initial corn steep liquor concentration was increased by 40%, the potency of spiramycin was further improved and the impurity D was further reduced. The representative results showed that after the initial corn steep liquor concentration was increased by 40%, the potency was increased by about 13.5% compared with the corresponding baseline, and the impurity D was reduced by about 11.0%. However, the impurity F may fluctuate slightly, suggesting that corn steep liquor fortification needs to be coordinated with the reduction of inorganic ammonium salts to avoid the potential risks brought about by a single measure.

[0050] Example 3: Reduction of initial ammonium salt dosage

[0051] Based on the addition of feed to remove soybean meal and the initial corn steep liquor increased by 40%, the amount of inorganic ammonium salt in the initial culture medium was reduced by 30% (i.e., 0.70 times the amount used in the original process). The purpose of this treatment is to reduce the excessive supply of readily available nitrogen sources in the early stage of fermentation, so that the cells can switch from utilizing readily available inorganic nitrogen sources to utilizing complex organic nitrogen sources earlier, and promote the pH recovery earlier.

[0052] The results are as follows Figure 4 As shown, after reducing the initial ammonium salt content by 30%, the pH recovery during fermentation was 12 hours earlier, indicating an earlier initiation time for secondary metabolism. Simultaneously, potency was further improved, and both impurity D and impurity F decreased. Representative results indicate that this treatment further increased potency by approximately 5.5% compared to the corresponding baseline, reduced impurity F by approximately 14.6%, and reduced impurity D by approximately 11%. These results confirm the synergistic effect of ammonium salt reduction and corn steep liquor enhancement; the combination of these two measures can achieve better control of nitrogen metabolism rhythm.

[0053] Example 4: Comprehensive process optimization (initially increasing corn steep liquor + reducing ammonium salts)

[0054] In a 15L fermenter, a comprehensive optimized process was adopted, which included feeding to remove soybean meal, increasing the initial corn steep liquor by 40%, and reducing the initial ammonium salt by 30%. The original process was used as a control to verify the complete fermentation cycle.

[0055] Valence results: by Figure 6 As shown in A, the potency of the conditional tank was higher than that of the control tank throughout the fermentation process. At 120 h, the potency of the control tank was 16693 U / mL, while that of the conditional tank was 20927 U / mL, which was about 25.4% higher than that of the control. This indicates that the process of adding corn steep liquor to reduce ammonium salt can significantly promote the accumulation of spiramycin.

[0056] Impurity F control: by Figure 6 As shown in B, the difference in the proportion of impurity F between the two groups is small, and the endpoint condition tank is slightly lower than the control tank, indicating that the process does not significantly increase the risk of impurity F, and the comprehensive regulation effectively balances the potential impact of corn steep liquor fortification.

[0057] Impurity D control: by Figure 6 As shown in Figure C, the impurity D in the condition tank was significantly lower than that in the control tank. At 120 h, the impurity D in the control tank was approximately 5.53%, while that in the condition tank was approximately 3.61%, a reduction of about 34.7%. This result indicates that the corn steep liquor reduction process has a significant effect on controlling impurity D, which may be related to the optimization of nitrogen metabolism rhythm and the redistribution of carbon metabolism flux.

[0058] Fermentation process stability: by Figure 6As can be seen from the DF data, the trends of total sugar, amino nitrogen, and bacterial concentration in the two groups are generally similar, indicating that the process did not cause obvious fermentation abnormalities and has good process stability and operability.

[0059] Overall conclusion: Adding corn steep liquor and reducing ammonium salts after removing soybean meal from the feed can increase the spiramycin potency by about 25.4% and reduce impurity D by about 34.7%, while keeping impurity F basically stable. This is the nitrogen source structure optimization process with better overall effect in this study.

[0060] Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.

[0061] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0062] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A nitrogen-source-controlled fermentation method for spiramycin, characterized in that, Includes the following steps: A1. Preparation of initial culture medium: Based on the existing spiramycin fermentation culture medium, increase the amount of corn steep liquor to 1.25 to 1.40 times the original amount, and reduce the amount of inorganic ammonium salt to 0.60 to 0.80 times the original amount; A2. Preparation of supplemental culture medium: Eliminate or significantly reduce the amount of soybean meal powder added; A3. Control the fermentation process: Monitor pH, amino nitrogen, total sugar, cell concentration, spiramycin potency, impurity D and impurity F during the fermentation process, and use pH rise and amino nitrogen change as process judgment indicators of nitrogen source utilization rhythm and secondary metabolism initiation state. A4. Control fermentation time: The fermentation cycle is 120 hours. After fermentation, the spiramycin potency and impurity composition are tested.

2. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, The amount of corn steep liquor used in A1 is 1.40 times that of the original process.

3. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, The amount of inorganic ammonium salt used in A1 is 0.70 times that of the original process.

4. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, Soybean meal powder is not added to the feed medium described in A2.

5. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, The inorganic ammonium salt includes ammonium sulfate, ammonium chloride, or mixtures thereof.

6. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, The fermentation process uses Streptomyces disacchari as the production strain.

7. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, Also includes: The expression levels of genes related to spiramycin biosynthesis during fermentation were monitored to verify the mechanism by which nitrogen source regulation affects secondary metabolism.

8. The spiramycin fermentation method based on nitrogen source regulation according to claim 7, characterized in that, The biosynthesis-related genes include macrocyclic lactone backbone synthesis genes, deoxyglycosylation genes, and glycosyltransferase genes.

9. The spiramycin fermentation method based on nitrogen source regulation according to claim 1, characterized in that, The pH recovery time is 10 to 15 hours in advance.

Images