Method for promoting erythromycin fermentation production by combined addition of vitamins

Abstract

The erythromycin fermentation culture medium is optimized by comparing transcriptomics analysis. Specifically, the invention relates to a method for optimizing the fermentation yield of erythromycin by adding six vitamins into a culture medium in a combined manner. More specifically, the six vitamins comprise thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin and hemin. The vitamin is added into the fermentation culture medium, so that the growth of erythromycin producing bacteria in the fermentation culture medium is promoted, and the yield of erythromycin is effectively increased.

Description

Technical Field

[0001] The present invention belongs to the field of biological fermentation technology, and more specifically, the present invention relates to a method for optimizing erythromycin fermentation medium by adding vitamins in combination based on comparative transcriptome analysis. Background Technology

[0002] As a typical macrolide antibiotic, erythromycin has the characteristics of broad-spectrum anti-Gram-positive bacteria and is widely used in the medical industry and agriculture. Exploring the high-yield mechanism of erythromycin in S.erythraea and continuously optimizing and upgrading its fermentation process are of great significance and reference value for the industrial production of erythromycin and the development of new natural products. With the rapid development of nucleic acid sequencing technology, comparative transcriptomics technology has gradually been applied to the research of discovering and improving the synthesis of secondary metabolites by industrial microorganisms, and can comprehensively and effectively explore the key factors affecting the production of secondary metabolites. Previously, comparative transcriptomics technology has been rarely used in the optimization of fermentation medium.

[0003] Vitamins are cofactors of many key catalytic enzymes in organisms. Enhancing their intracellular supply can effectively enhance enzyme activity and promote bacterial growth and metabolic activity. In actinomycete systems, vitamins have also been shown to have a significant effect on the synthesis of secondary metabolites. However, there are few reports on the application of multiple vitamin combinations in optimizing erythromycin fermentation medium. SUMMARY OF THE INVENTION

[0004] In view of the shortcomings of the prior art, the present invention provides a method for optimizing the fermentation medium of erythromycin produced by Saccharopolyspora erythropolis based on comparative transcriptomics analysis. By comparing the gene levels of the erythromycin high-producing strain HL3168-E3 and the erythromycin low-producing standard strain NRRL23338 in the growth stage and the erythromycin production stage, it was found that the transcription levels of B vitamin synthesis genes were collectively upregulated in the erythromycin production stage of the erythromycin high-producing strain. By adding vitamins separately to the fermentation medium, six vitamins were determined: thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin and hemin, which have a promoting effect on the synthesis of erythromycin. Through the biostatistics method, the inventor optimized the ratio of the six vitamin combinations added and found that adding the six vitamin combinations to the fermentation medium can significantly increase the yield of erythromycin.

[0005] In a first aspect of the present invention, a vitamin combination for erythromycin fermentation in a synthetic medium is provided, wherein the vitamin combination comprises the following components: thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin and hemin.

[0006] In a preferred embodiment, the combination of vitamins is thiamine pyrophosphate at 0.1 - 10 mg / L, riboflavin at 0.1 - 10 mg / L, pyridoxine at 0.1 - 10 mg / L, folic acid at 0.1 - 10 mg / L, cobalamin at 0.1 - 10 mg / L, and hemin chloride at 0.1 - 10 mg / L.

[0007] In another preferred embodiment, the combination of vitamins is thiamine pyrophosphate at 0.1 - 1 mg / L, riboflavin at 0.1 - 1 mg / L, pyridoxine at 0.1 - 1 mg / L, folic acid at 1 - 10 mg / L, cobalamin at 0.1 - 1 mg / L, and hemin chloride at 1 - 10 mg / L

[0008] In another preferred embodiment, thiamine pyrophosphate is at 0.5 mg / L, riboflavin is at 0.15 mg / L, pyridoxine is at 0.21 mg / L, folic acid is at 5 mg / L, cobalamin is at 0.39 mg / L, and hemin chloride is at 4 mg / L

[0009] Therefore, in one aspect, the present patent disclosure provides a method for producing erythromycin by fermentation in a synthetic medium, comprising adding a total of six vitamins, namely thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin, and hemin chloride, to the fermentation medium.

[0010] In another aspect, the present patent disclosure provides the use of a combination of six vitamins, namely thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin, and hemin chloride, for increasing the fermentation yield of erythromycin.

[0011] In another aspect, the thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin, and hemin chloride described in the present patent include their acid, base, and salt forms.

[0012] The addition of the combination of thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin, and hemin chloride can increase the erythromycin titer in the fermentation broth at the end of fermentation. Compared with the control fermentation of a synthetic medium without any vitamin addition, the increase in this titer can reach at least 15%, preferably at least 20%, at least 25%, at least 30%, or at least 35%.

[0013] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following, in conjunction with the drawings, through a detailed description of the specific embodiments of the present application, will make the technical solutions and other beneficial effects of the present application obvious.

[0015] Figure 1 This is the gene transcriptome comparison analysis and classification results carried out using the erythromycin high-yield strain HL3168-E3 and the Saccharopolyspora erythraea standard strain NRRL2333 in the examples of the present application.

[0016] Figure 2 This is the result graph for exploring the effect of single vitamin gradient addition on erythromycin synthesis in the embodiments of this application.

[0017] Figure 3 This is the result response value when combined vitamins are added in the Plackett - Burman experiment in the embodiments of this application.

[0018] Figure 4 This is the result of verifying the influence of vitamin addition on the macroscopic metabolic parameters of erythromycin fermentation using a 5L fermenter in the embodiments of this application. Detailed implementation manners

[0019] Next, the technical solutions in the embodiments of this application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. The present invention covers all possible alternative solutions, improvement solutions, and equivalent solutions within the scope of the claims. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of this application.

[0020] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for illustrative purposes and do not limit the content of this application. The specific technologies or conditions not specified in the following embodiments are all conventional technologies or conditions, or are in accordance with the technologies or conditions described in the literature in this field, or are in accordance with the product instructions.

[0021] I. The formulation and preparation of the culture medium involved in the embodiments of this application are as follows:

[0022] (1) Slant medium

[0023] In every 100 mL of slant medium, it includes: 1.2 g of corn steep liquor, 1.0 g of starch, 0.3 g of NaCl, 0.3 g of (NH 4 ) 2 SO 4 , 0.5 g of CaCO 3 , and 2.0 g of agar.

[0024] Preparation of 100 mL slant medium: First, weigh each component according to the formula; then, dissolve each component separately in deionized water, make up the volume to 100 mL, and adjust the pH to 7.0 with NaOH; next, place the prepared medium in an autoclave and autoclave at 121 °C for 15 min; finally, dispense the sterilized medium into multiple test tubes and perform slant setting to obtain the slant medium.

[0025] (2) Seed medium

[0026] In every 100 mL of seed medium, it includes: 2.0 g of peptone, 4.0 g of starch, 2.0 g of dextrin, 0.4 g of NaCl, 0.02 g of KH 2 PO 4 , 0.025 g of MgSO 4 , 0.6 g of soybean oil and 0.6 g of CaCO 3 .

[0027] Preparation of 100 mL seed medium: First, weigh each component according to the formula; then, dissolve each component separately in deionized water, make up the volume to 100 mL, and adjust the pH to 7.0 with NaOH; next, place the prepared medium in an autoclave and autoclave at 121 °C for 15 min; finally, add glucose to the sterilized medium, and the final concentration of glucose in the medium is 1 g / mL to obtain the seed medium.

[0028] (3) Basic medium

[0029] In every 800 mL of basic medium, it includes: 20.0 g of (NH 4 ) 2 SO 4 , 50.0 g of casein hydrolysate, 0.6 g of MgSO 4 ·7H 2 O, 1.0 g of ZnSO 4 ·7H 2 O, 1.0 g of FeSO 4 ·7H 2 O, 1.0 g of MnCl 2 ·4H 2 O and 1.0 g of CaCl 2 .

[0030] Preparation of 800 mL basic medium: First, weigh each component according to the formula; then, dissolve each component separately in deionized water, make up the volume to 800 mL; next, place the prepared medium in an autoclave and autoclave at 121 °C for 15 min; then, add 150 mL of NaH 2PO 4 / K 2 HPO 4 Buffer solution (concentration: 0.1 mol / L, pH = 6.8); finally, add glucose with a weight - volume ratio of 25% to the culture medium, and the final concentration of glucose in the culture medium is 20 g / L to prepare the basal culture medium.

[0031] (4) Fermentation synthetic medium

[0032] In every 100 mL of fermentation medium, it includes: 2 g of glucose, 0.128 g of K 2 HPO 4 , 0.064 g of KH 2 PO 4 , 0.1 g of MgSO 4 ·7H 2 O, 0.086 g of alanine, 0.068 g of arginine, 0.078 g of cysteine, 0.073 g of serine and 0.1 mL of 100× trace elements.

[0033] Preparation of 100 mL of synthetic fermentation medium: First, weigh each component according to the formula; then, dissolve each component separately in deionized water, make up the volume to 100 mL, and adjust the pH = 7.0 with HCl. Next, place the prepared medium in an autoclave and sterilize it at 121 °C for 20 min; finally, add glucose with a weight - volume ratio of 20% to the medium, and the final concentration of glucose in the medium is 20 g / L to prepare the synthetic fermentation medium.

[0034] Second, the cultivation method in the examples of this application is as follows:

[0035] Cultivation method - slant culture:

[0036] Take out the strain preserved in a glycerol tube from the - 80 °C refrigerator, pipette 50 μL and spread it on a plate, and culture it in a constant - temperature incubator at 34 °C for 4 - 6 days

[0037] Cultivation method - seed flask culture:

[0038] Take out the plate from the 34 °C constant - temperature incubator, use an inoculation spatula to dig a 1 - square - centimeter piece and inoculate it into the shake - flask medium, and culture it at 34 °C and 220 rpm for 48 hours

[0039] Cultivation method - 500 mL shake - flask fermentation

[0040] Pour 50 mL of the bacterial liquid in the seed flask into a 50 mL graduated sterile centrifuge tube in a laminar flow hood, and centrifuge at 6000 rpm for 3 min at room temperature; discard the supernatant completely in the laminar flow hood, add 20 mL of sterile deionized water for resuspension, and centrifuge at 6000 rpm for 3 min at room temperature; discard the supernatant completely in the laminar flow hood, add about 20 mL of sterile deionized water to make up to 25 mL, pipette and resuspend with a 5 mL pipette, and the bacterial suspension can be used for inoculating the synthetic fermentation medium, with an inoculation amount of 2.5 mL. After inoculation, place the flask in an incubator at 34 °C and 220 rpm, and sample and detect the offline parameters at intervals of 12 or 24 h.

[0041] Cultivation method - fermentation in a 5 L bioreactor

[0042] The filling volume of the synthetic medium in the 5 L bioreactor is 3 L, and 0.05%-0.1% of polyether defoamer needs to be added additionally during the preparation of the medium. The fermentation seeds are washed in the same way as in flask fermentation and inoculated by flame, with an inoculation amount of 300 mL. The initial stirring speed of the reactor is set at 300 rpm, the aeration rate is set at 1 vvm, the culture temperature is set at 34 °C, the tank pressure is set at 0.03 mPa, and the dissolved oxygen in the system is controlled not to be lower than 30% of the critical dissolved oxygen through the speed linkage control during the fermentation process.

[0043] III. Reagent description involved in the embodiments of the present application

[0044] The reagents involved in the embodiments of the present application, including: molecular biology reagents, chemical reagents, vitamins (such as riboflavin, pyridoxine, folic acid, cobalamin) and antibiotics (such as: erythromycin, ampicillin, apramycin, etc.), are all commercial products and are available on the market.

[0045] The transcriptome sequencing involved in the embodiments of the present application was completed by Majorbio Co., Ltd.

[0046] The following is a detailed description of the embodiments of the present application. This embodiment is implemented on the premise of the technical solution of the present application, and the detailed implementation method and specific operation process are given.

[0047] Example 1

[0048] Comparative transcriptome analysis of high erythromycin-producing strain HL3168-E3 and the standard strain of Saccharopolyspora erythraea NRRL23338

[0049] To analyze the high-yield mechanism of erythromycin in the logarithmic phase of E3 at the transcriptional level, RNA-seq technology was used to characterize the transcriptional profiles of WT and E3 in different metabolic states. According to the growth curve and product accumulation curve, the logarithmic growth phase (10 h) and stationary phase (50 h) were selected as the sampling points for comparative transcriptomics analysis. The collected samples were entrusted to Majorbio Co., Ltd. for comparative transcriptomics analysis. Using the expression quantification software RSEM, the expression levels of genes in the 4 groups of samples were quantitatively analyzed respectively, and the quantitative index was (Fragments Per Kilobase of exon per Million mapped reads) FPKM. After obtaining the relative quantitative data of the expression levels, DESeq2 was used for differential gene expression analysis between groups, and different significantly differentially expressed genes between groups were screened out. During the analysis process, the default was to use the fold change of up / down-regulation greater than 2 and the p-value less than 0.05 as the threshold for screening differential genes, and the results of differential expression analysis were characterized by drawing volcano plots. To identify the specifically up-regulated and down-regulated genes between different groups, the up-regulated and down-regulated genes under different alignment conditions were set as different gene sets respectively, and the common and unique genes in each gene set were identified through Venn analysis.

[0050] Among the up-regulated genes under the 4 alignment conditions, the total number of genes up-regulated in E3 compared with WT at 50 h was 2168, and the number of specifically up-regulated genes was 1060. The total number of genes up-regulated in E3 at 50 h compared with 10 h was 1107, and the number of specifically up-regulated genes was 477. The number of genes that were up-regulated genes under both of these two alignment conditions was 337. Summarizing the above three groups of up-regulated genes, they jointly constituted the gene set positively correlated with erythromycin synthesis, and this gene set contained a total of 1874 genes. Given that the high-yield strain showed a high erythromycin synthesis ability during the production period, the specific up-regulation of the transcription of genes in this gene set was considered to be beneficial to secondary metabolism. Based on the same analysis idea, a gene set negatively correlated with erythromycin synthesis was constructed. The down-regulation of the expression of these genes was considered to be beneficial to the synthesis of erythromycin, and this gene set contained a total of 2035 genes.

[0051] The genes in the above gene sets positively and negatively correlated with erythromycin synthesis were respectively subjected to KEGG functional annotation analysis, and the Figure 1 results were obtained. The up-regulated and down-regulated genes were distributed in a relatively high proportion in the cell metabolic pathway, and there were more differentially expressed genes in KEGG pathways such as carbon metabolism, amino acid metabolism, cofactor and vitamin metabolism, lipid metabolism and energy metabolism. Among them, the number of genes with up-regulated expression in the cofactor and vitamin metabolism and energy metabolism pathways was significantly higher than the number of genes with down-regulated expression, indicating that the overall expression level of genes in these pathways was enhanced during the erythromycin-producing period of E3, which might be beneficial to the synthesis of erythromycin.

[0052] To more intuitively understand the distribution of differentially expressed genes in the vitamin synthesis and metabolism pathways, the transcriptional levels of related genes in different groups were normalized and classified according to the vitamin synthesis pathways. The results of transcriptional analysis showed that the genes involved in the synthesis and metabolism of most B vitamins had higher expression levels in the E3 stationary phase than in the E3 growth phase or the WT stationary phase. Considering that vitamins are important sources of various cofactors in cells, the changes in the expression of genes in these related pathways may affect the concentrations of intracellular vitamins and cofactors, and thus change the growth and secondary metabolism-related characteristics of S. erythraea.

[0053] Example 2

[0054] Single vitamin gradient addition experiment in the erythromycin synthesis fermentation medium

[0055] First, the single vitamin addition experiment was carried out. The experiment involved 9 different vitamins or their derivatives, including thiamine pyrophosphate TPP, riboflavin VB2, niacin VB3, pantothenic acid VB5, pyridoxine VB6, biotin VB7, folic acid VB9, cobalamin VB12, and hemin. The addition of vitamins was carried out at the initial stage of fermentation in the synthetic medium. The results of the experiment are as Figure 2 shown

[0056] Considering that some vitamins are unstable at high temperatures, the vitamin solutions were sterilized by filtration through a 0.2 μM pore size filter head during the addition experiment. The vitamin solutions involved in the present invention were all freshly prepared before the addition experiment.

[0057] Example 3

[0058] Plackett - Burman experiment

[0059] To explore the comprehensive effects of the combined addition of multiple vitamins on the growth of E3 and erythromycin synthesis and to identify the optimal vitamin addition combination, the present invention carried out an experiment on the combined addition and concentration optimization of multiple vitamins. Six vitamins that promoted erythromycin synthesis in Example 2 were selected for the Plackett - Burman (PB) experiment to explore the main factors affecting erythromycin synthesis. The high level was set to be 3 times the low level in the experiment, and a total of 13 experiments were carried out. The design of the PB experiment is shown in Table 1, the independent variables, coding, and level factors are shown in Table 2, and the results of the PB experiment are shown in Table 3 and Figure 3 shown.

[0060] Table 1. Factor levels of the Plackett - Burman experiment.

[0061]

[0062] Table 2, Plackett-Burman experimental design table.

[0063] RUN F1 F2 F3 F4 F5 F6 1 -1 -1 -1 1 -1 1 2 1 1 1 -1 -1 -1 3 -1 -1 1 1 1 1 4 1 -1 1 1 1 -1 5 -1 1 1 1 -1 -1 6 -1 -1 -1 1 -1 -1 7 1 -1 1 1 -1 1 8 1 1 -1 1 -1 1 9 1 1 -1 1 1 1 10 -1 1 -1 1 1 -1 11 1 -1 1 1 1 -1 12 0 0 0 0 0 0 13 -1 1 1 1 1 1

[0064] Table 3, Analysis of variance of Plackett–Burman (PB) experimental model.

[0065]

[0066] Example 4

[0067] Steepest ascent experiment

[0068] In order to further optimize the composition of vitamin addition, three factors that have the greatest impact on erythromycin biosynthesis were selected for the steepest ascent experiment, namely vitamin B 2 , vitamin B 6 , and vitamin B 12 . According to the estimated coefficient model obtained in the PB experiment, for the three components of vitamin B 2 , vitamin B 6 , and vitamin B 12 , the positive and negative effects of the estimated coefficients of these three components were increased or decreased in turn. Among them, vitamin B 12 has a positive effect and should be increased in turn, while vitamin B 2 and vitamin B 6 have negative effects and should be decreased in turn. The design and results of the steepest ascent experiment are shown in Table 4.

[0069] Table 4, Design and results of the steepest ascent experiment

[0070]

[0071] Example 5

[0072] In order to explore the effect of vitamin addition on the macroscopic metabolic characteristics of E3, the present invention carried out vitamin combination addition experiments in a 5L bioreactor, detected the corresponding offline and online parameters, and verified the effect of vitamin addition on cell growth and erythromycin synthesis.

[0073] The liquid loading of the synthetic medium in the 5L bioreactor is 3L, and 0.05%-0.1% of polyether defoamer needs to be added additionally during the preparation of the medium. The fermentation seeds were washed and flame-inoculated in the same way as in shake flask fermentation, and the inoculation amount was 300 mL. The initial stirring speed of the reactor was set at 300 rpm, the aeration rate was set at 1 vvm, the culture temperature was set at 34 °C, the tank pressure was set at 0.03 mPa, and the dissolved oxygen in the system was controlled not to be lower than 30% of the critical dissolved oxygen through the speed linkage control during the fermentation process. The results of the fermentation experiment carried out in the 5L bioreactor are asFigure 4 As shown, it can be seen that adding the vitamin combination of the present invention can promote the early growth of the bacterial cells and significantly increase the fermentation yield of erythromycin.

[0074] The above has introduced in detail a certain one provided by the embodiments of the present application. Specific examples are used herein to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the technical solution and its core idea of the present application; those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for producing erythromycin by fermentation by optimizing fermentation medium, characterized in that One or more vitamin combinations are added to the erythromycin fermentation medium.

2. The method of the preceding claim, wherein the types of vitamins include thiamine pyrophosphate, niacin, pantothenic acid, riboflavin, pyridoxine, folic acid, cobalamin and hemin, etc.; wherein the types of vitamins may further preferably be thiamine pyrophosphate, riboflavin, pyridoxine, folic acid, cobalamin and hemin.

3. The method of the preceding claim, wherein the amount of the vitamins used can further preferably be: thiamine pyrophosphate 0.1-10 mg / L, riboflavin 0.1-10 mg / L, pyridoxine 0.1-10 mg / L, folic acid 0.1-10 mg / L, cobalamin 0.1-10 mg / L and hemin 0.1-10 mg / L.

4. The method of the preceding claim, wherein the amount of the vitamins used can further preferably be: thiamine pyrophosphate 0.1-1 mg / L, riboflavin 0.1-1 mg / L, pyridoxine 0.1-1 mg / L, folic acid 1-10 mg / L, cobalamin 0.1-1 mg / L and hemin 1-10 mg / L.

5. The method of the preceding claim, wherein the amounts of the vitamins used may further preferably be: thiamine pyrophosphate 0.5 mg / L, riboflavin 0.15 mg / L, pyridoxine 0.21 mg / L, folic acid 5 mg / L, cobalamin 0.39 mg / L and hemin 4 mg / L.

6. The method of the preceding claim, wherein the vitamin comprises its acid, base and salt forms.

7. The process of the preceding claims, wherein the vitamin combination is used as the total vitamin addition to the fermentation medium, or is used in combination with other vitamins.

8. The process of the preceding claim, wherein the vitamins are added in combination between 0 and 48 hours, preferably between 0 and 24 hours, of the fermentation.

9. The method of the preceding claim, wherein the fermentation is carried out at a pH of from 6.0 to 8.5, such as from pH 7.0 to 7.

5.

10. The process of the preceding claim, wherein the fermentation is carried out at a fermentation temperature of about 25°C to 40°C, preferably about 28-35°C, more preferably about 34°C.

11. The process of the preceding claim, wherein the fermentation medium comprises a carbon source, a nitrogen source and salt ions, and optionally one or more other components selected from trace elements, pH regulators and antifoaming agents.

12. The method of the preceding claim, wherein the fermentation medium comprises a carbon source, wherein the carbon source comprises sugars and sugar-containing Material; preferably, the synthetic medium comprises a carbon source selected from monosaccharides, disaccharides and combinations thereof; more preferably, the fermentation medium comprises a carbon source selected from glucose, sucrose and combinations thereof, or the fermentation medium comprises only a single carbon source such as glucose.

13. The method of the preceding claim, wherein the synthetic culture medium comprises a nitrogen source, wherein the nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source; preferably, the fermentation medium comprises one or more nitrogen sources selected from the following: soybean cake powder, corn steep liquor, amino acids, ammonia, ammonium, ammonium salts, urea, nitric acid, nitrates and combinations thereof; more preferably, the fermentation medium comprises one or more nitrogen sources selected from the following: soybean cake powder, corn steep liquor, ammonium sulfate, ammonium citrate, glutamic acid, cysteine, serine, arginine, aspartic acid, proline and combinations thereof.

14. The method of the preceding claim, wherein the fermentation medium comprises trace elements, preferably at least one trace element selected from the group consisting of Co, Cu, Mo, Mn, Zn, Fe, borates, more preferably CuCl2, (NH4)6Mo7O 24 , one or more of CoCl2, Na2B4O7 and FeCl3.

15. The process of the preceding claim, wherein the fermentation medium comprises an erythromycin producing strain, such as Saccharopolyspora cells, preferably Saccharopolyspora erythrae cells.

16. A kit for optimizing erythromycin fermentation production, characterized in that: The kit comprises the vitamin combination according to any one of claims 1 to 6.

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