Culture medium for biosynthesis of indigoidine and application thereof

Abstract

This invention discloses a culture medium for the biosynthesis of indigoidine and its application, belonging to the field of microbial fermentation and synthetic biology technology. Addressing the problems of low yield, insufficient precursor supply, byproduct accumulation, and poor scale-up stability in existing culture media for indigoidine fermentation, this invention provides a core culture medium system. This medium, per 1L, includes: 10-50g carbon source, 5-20g nitrogen source, 10-30g L-glutamine, and 0.1-20g inorganic salts, with optional addition of 0.1-20mL trace element salt solution and / or vitamin components. The pH is 6.0-7.5. The carbon source is selected from glucose, glycerol, sucrose, dextrin, or combinations thereof; the nitrogen source is selected from ammonium sulfate, yeast extract, peptone, or combinations thereof. This culture medium can significantly increase the yield of indigoidine synthesized by recombinant *E. coli* and effectively inhibit acetic acid accumulation. In a 5L fermenter, compared with LB medium, this medium can increase the indigoidine yield by at least 80% while reducing the acetic acid concentration in the fermentation broth by more than 60%, showing good prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation, synthetic biology and natural pigment manufacturing technology, and in particular to a culture medium for the biosynthesis of Indigoidine and its application. Background Technology

[0002] Indigoidine, a bright, natural blue pigment with antioxidant and antibacterial activities, has broad application prospects in the food, cosmetics, textile, and pharmaceutical industries. Due to the scarcity of natural blue pigments, green preparation using microbial fermentation methods has significant industrial value.

[0003] Currently, recombinant E. coli heterologously expressing the Indigoidine synthase gene is commonly used to produce Indigoidine. However, existing processes generally use LB medium or simple synthetic medium, which face the following key bottlenecks in industrial production: 1. Low yield, making it difficult to meet the economic requirements of industrialization; 2. Insufficient precursor supply, as the biosynthesis of Indigoidine strongly depends on precursors such as L-glutamine, which conventional mediums cannot provide a sufficient and balanced supply; 3. Byproduct accumulation, as metabolic byproducts such as acetic acid easily accumulate in the later stages of fermentation, inhibiting cell growth and product synthesis; 4. Significant scale-up effect, as optimized process parameters in the shake flask stage are difficult to reproduce in the fermenter, leading to decreased yield and stability; 5. High cost, as existing high-yield schemes often use expensive organic nitrogen sources or special components, which are not conducive to large-scale application.

[0004] Therefore, developing a dedicated Indigoidine culture medium system and supporting processes that can achieve high yield, stability, low by-products, easy scale-up, and controllable cost is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for the biosynthesis of Indigoidine and its application. This culture medium, through systematic optimization of the composition and ratio of carbon source, nitrogen source, precursor, inorganic salt and trace elements, aims to adapt to the biosynthetic pathway of Indigoidine, thereby significantly increasing its yield, inhibiting the generation of harmful byproducts, and improving the stability of the process.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A culture medium for the biosynthesis of indigoidine, characterized in that, per 1L, it comprises the following components: 10-50g carbon source, 5-20g nitrogen source, 10-30g L-glutamine, 0.1-20g inorganic salt, optionally with the addition of 0.1-20mL trace element salt solution and / or vitamin components, with the remainder being water; the pH of the culture medium is 6.0-7.5; the carbon source is one or a combination of two or more of glucose, glycerol, sucrose, and dextrin; and the nitrogen source is one or a combination of two or more of ammonium sulfate, yeast extract, and peptone.

[0007] Preferably, the inorganic salt includes one or more of KH2PO4, K2HPO4, MgSO4, CaCl2, and NaCl.

[0008] Preferably, the trace element salt solution contains Zn. 2＋ Fe 3＋ Cu 2＋ Mn 2＋ Mo 6＋ B 3＋ At least one of them.

[0009] Preferably, the vitamin components include one or more of inositol, calcium pantothenate, niacin, para-aminobenzoic acid, pyridoxine hydrochloride, and riboflavin.

[0010] As a preferred option, the culture medium is suitable for shake flask fermentation and contains, per 1L: 8-12g glucose, 3-8g [(NH4)2SO4], 1-3g KH2PO4, 1-3g K2HPO4, 10-25g L-glutamine, 8-12g glycerol, 8-12mL trace element salt solution, and pH 7.0-7.2.

[0011] As a preferred option, the culture medium is suitable for fermentation in a fermenter and contains, per 1L: 8-12g glucose, 7-8g [(NH4)2SO4], 1-3g KH2PO4, 2-3g K2HPO4, 20-25g L-glutamine, 8-12g glycerol, 8-12mL trace element salt solution, and pH 6.8-7.2.

[0012] The use of any of the above-mentioned culture media in the preparation of Indigoidine.

[0013] Preferably, the application is in the cultivation of recombinant Escherichia coli expressing Indigoidine synthase to produce Indigoidine.

[0014] The beneficial effects of this invention are as follows: High-yield characteristics and metabolic regulation advantages: By optimizing the dual carbon source combination of glucose and glycerol, and providing sufficient and balanced ammonium sulfate as a nitrogen source and L-glutamine as a precursor, a highly efficient metabolic pathway adapted to the synthesis of indigoidine is constructed. More importantly, by introducing a precisely proportioned phosphate buffer system, the pH of the fermentation environment is effectively stabilized, and the cellular metabolic flux is synergistically optimized. Experiments have shown that, using the medium of this invention, in a 5L fermenter, the yield of indigoidine can be increased by more than 80% compared with the traditional LB medium, while the acetic acid concentration in the fermentation broth can be reduced by more than 60%, significantly improving production efficiency and product purity.

[0015] Excellent scale-up stability: This invention optimizes the ratio of phosphate and ammonium sulfate for the different process characteristics of shake flasks and fermenters, so that the culture medium provides sufficient nutrition while having good pH buffering capacity and osmotic pressure adaptability. This design ensures process stability and yield consistency from laboratory pilot to pilot-scale scale-up, effectively overcoming the scale-up effect.

[0016] Clear composition and economy: The main components of the culture medium are clear chemical reagents or food-grade raw materials, avoiding the use of organic nitrogen sources with complex composition and large batch-to-batch variation (such as soybean flour). This not only ensures the controllability and reproducibility of the fermentation process, but also reduces the difficulty and cost of subsequent product separation and purification, which is more conducive to industrial production. Attached Figure Description

[0017] Figure 1 A comparison of the yield of Indigoidine in shake-flask fermentation using the preferred culture medium and the conventional LB medium; Figure 2 The graph shows the changes in Indigoidine yield versus acetic acid concentration during the fermentation process of the preferred culture medium in a 5L fermenter according to the present invention. Detailed Implementation

[0018] The following description is only a preferred embodiment of the present invention, and the scope of protection is not limited to this embodiment. Any technical solution that falls within the scope of the present invention should be protected by the present invention.

[0019] Comparative Example 1 Traditional LB medium fermentation Prepare standard LB liquid medium: Inoculate the preserved engineered strain onto LB agar plates containing 50 µg / mL kanamycin for activation. Pick a single colony and inoculate it into 5 mL of LB seed culture (containing kanamycin). Incubate overnight at 37°C with shaking at 220 rpm. Transfer 2% of the inoculum to 50 mL of fermentation medium (250 mL shake flask) and incubate at 37°C until OD reaches 0.50. 600Approximately 0.6-0.8 mg / L of IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25°C for 24 hours. After fermentation, samples were taken to determine the indigoidine yield and acetic acid concentration. The results are as follows: Figure 1 As shown, the yield of Indigoidine in LB medium was the baseline (set to 100%), and the acetic acid concentration at the fermentation endpoint was relatively high.

[0020] Example 1 Culture medium for shake-flask fermentation and its effects The shake-flask fermentation medium of this invention comprises, per liter: 10.0 g glucose, 10.0 g glycerol, 5.0 g ammonium sulfate [(NH4)2SO4], 2.0 g KH2PO4, 2.0 g K2HPO4, 20.0 g L-glutamine, and a trace element salt solution (containing ZnCl2, FeCl3·6H2O, CuCl2, MnCl2·4H2O, Na2B4O7·10H2O, (NH4)6Mo7O) 24 Add 10 mL of NaOH solution (such as 4H2O) and adjust the pH to 7.0. Make up to 1 L and sterilize at 121°C for 20 minutes.

[0021] Using the same recombinant Escherichia coli strain and fermentation process as Comparative Example 1 (seed liquid preparation, inoculum size, and induction conditions were all the same), three parallel experiments were set up. Shake-flask fermentation was carried out in this culture medium. After fermentation, the indigoidine yield of the three parallel samples was measured and the mean and relative standard deviation (RSD) were calculated. At the same time, the acetic acid concentration was measured.

[0022] Results (e.g.) Figure 1 As shown): The average yield of indigoidine in the culture medium of this example reached 2.5 times that of Comparative Example 1 (LB medium), and the RSD was less than 5%, showing good reproducibility. The concentration of acetic acid in the fermentation broth was reduced by about 40% compared with Comparative Example 1.

[0023] Example 2 Fermentation culture medium and its effects in fermentation tanks The fermentation medium for the fermenter of this invention is prepared as follows: per liter, it contains: 10.0 g glucose, 10.0 g glycerol, 7.6 g ammonium sulfate [(NH4)2SO4], 2.0 g KH2PO4, 2.5 g K2HPO4, 23.4 g L-glutamine, and 10 mL of trace element salt solution as in Example 1, with a natural pH (approximately 6.9), and is subjected to actual digestion at 121°C for 30 minutes.

[0024] Fermentation was carried out in a 5L fermenter (working volume 3L). The prepared seed culture was inoculated into the fermenter at a rate of 5%. Initial culture conditions were: temperature 37℃, pH 6.9 (controlled by automatic ammonia addition), and dissolved oxygen (DO) maintained above 30% (by adjusting the stirring speed and aeration rate). When OD... 600 When the temperature reaches approximately 10°C, the temperature is lowered to 25°C, and IPTG is added to a final concentration of 0.5 mM for induction. After induction, the residual sugar concentration is maintained by feeding a fed broth (mainly containing glucose and glycerol). Samples are taken periodically throughout the fermentation process to monitor the OD (oxidative stress). 600 Indigoidine production and acetic acid concentration.

[0025] Results (e.g.) Figure 2 As shown): At the end of fermentation, the indigoidine yield was more than 3.0 times that of Comparative Example 1 (using LB medium in a shake flask). Throughout the fermentation process, the acetic acid concentration remained at a low level below 2 g / L, with the peak value being more than 60% lower than the fermentation endpoint of Comparative Example 1. This indicates that the medium of the present invention effectively regulates metabolism and significantly reduces the accumulation of harmful byproducts.

[0026] Example 3 L-glutamine concentration optimization experiment To verify the suitable concentration range of L-glutamine, based on the fermenter culture medium formulation of Example 2, other components were kept constant, and only the L-glutamine concentration was changed to 10 g / L, 20 g / L, 30 g / L, and 40 g / L for 5 L fermenter experiments, following the same procedure as in Example 2. Two parallel fermenters were set up for each concentration.

[0027] Results: When the L-glutamine concentration was 20-25 g / L, the production of indigoidine was the highest, and the cell growth (final OD) was also the highest. 600 The yield was good. When the concentration was increased to 30 g / L, there was no significant difference in yield compared to the 20-25 g / L group, but cell growth was slightly slowed. When the concentration reached 40 g / L, cell growth was significantly inhibited, and the final OD... 600 The yields of both L-glutamine and indigoidine decreased significantly. These experimental data support the scientific validity of setting the upper limit of L-glutamine at 30 g / L in claim 1, and indicate that 20-25 g / L is a more favorable range.

[0028] Example 4 Culture medium application Using the culture medium of Example 1 or Example 2, and following the corresponding fermentation process, recombinant Escherichia coli expressing Indigoidine synthase can be cultured efficiently to prepare Indigoidine. This method is suitable for the large-scale production of this natural blue pigment.

[0029] The specific embodiments described above further illustrate the technical problems, technical solutions, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for the biosynthesis of indigoidine, characterized in that, Based on 1L, the medium consists of the following components: 10-50g carbon source, 5-20g nitrogen source, 10-30g L-glutamine, 0.1-20g inorganic salt, optionally 0.1-20mL trace element salt solution and / or vitamin components, with the remainder being water. The pH of the culture medium is 6.0-7.

5. The carbon source is one or more of glucose, glycerol, sucrose, and dextrin, and the nitrogen source is one or more of ammonium sulfate, yeast extract, and peptone.

2. The culture medium according to claim 1, characterized in that, The inorganic salts include one or more of KH2PO4, K2HPO4, MgSO4, CaCl2, and NaCl.

3. The culture medium according to claim 1, characterized in that, The trace element salt solution contains Zn 2＋ Fe 3＋ Cu 2 ＋ Mn 2＋ Mo 6＋ B 3＋ At least one of them.

4. The culture medium according to claim 1, characterized in that, The vitamin components include one or more of inositol, calcium pantothenate, niacin, para-aminobenzoic acid, pyridoxine hydrochloride, and riboflavin.

5. The culture medium according to claim 1, characterized in that, It is a culture medium suitable for shake flask fermentation, containing, per 1L: 8-12g glucose, 3-8g [(NH4)2SO4], 1-3g KH2PO4, 1-3g K2HPO4, 10-25g L-glutamine, 8-12g glycerol, 8-12mL trace element salt solution, and pH 7.0-7.

2.

6. The culture medium according to claim 1, characterized in that, It is a culture medium suitable for fermentation in fermenters, containing, per 1L: 8-12g glucose, 7-8g [(NH4)2SO4], 1-3g KH2PO4, 2-3g K2HPO4, 20-25g L-glutamine, 8-12g glycerol, 8-12mL trace element salt solution, and pH 6.8-7.

2.

7. The use of the culture medium according to any one of claims 1-6 in the preparation of Indigoidine.

8. The application according to claim 7, characterized in that, The application is in the production of indigoidine by culturing recombinant Escherichia coli expressing indigoidine synthase.

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