Oregovomab-producing strain and application thereof

Abstract

This invention discloses a strain that produces an orivacin intermediate (A82846B) and its applications. The orivacin intermediate-producing strain, *Streptomyces desertica* JH100-32B16B, is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 17931. The fermentation method involves fermenting *Streptomyces desertica* CGMCC No. 17931 in a fermentation medium to produce the orivacin intermediate. Using this strain and its corresponding fermentation process, a fermentation potency of 1450 mg / L of A82846B can be achieved in a 30L fermenter using a low-cost fermentation medium, with fermentation costs reduced by more than 90% compared to the control process. Furthermore, the impurity-to-product ratio (A / B ratio) is as low as 1.5–2.0, more than half that of the control, which simplifies the product extraction process and reduces extraction costs. These two advantages combined result in a significantly lower overall production cost of A82846B compared to the control process reported in existing literature.

Description

Technical Field

[0001] This invention relates to the selection and fermentation technology of a microbial strain that produces a pharmaceutical intermediate, and particularly to the mutagenesis breeding of a microbial strain for producing the intermediate component A82846B of Orlistan and the corresponding fermentation process. Background Technology

[0002] Currently, in the field of antibacterial drugs, β-lactam broad-spectrum antibiotics, represented by penicillins and cephalosporins, are widely used in clinical practice. However, due to long-term, inappropriate use at high doses, a large number of drug-resistant pathogens have emerged in recent years and are constantly evolving, leading to traditional first-line antibiotics facing the predicament of poor or even ineffective antibacterial efficacy.

[0003] Glycopeptide antibiotics, due to their different mechanism of action compared to β-lactam antibiotics, exhibit excellent antibacterial effects against common β-lactamase-resistant bacteria. Vancomycin, as a representative of first-generation glycopeptide antibiotics, was used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections and once achieved excellent results. However, with its increasingly widespread clinical application, vancomycin-resistant Staphylococcus aureus (VRSA) and enterococci (VRE) have begun to emerge. First-generation glycopeptide antibiotics such as vancomycin and teicoplanin can no longer fully meet current clinical needs, necessitating the development of a new generation of glycopeptide antibiotics.

[0004] Oritavancin is a second-generation glycopeptide broad-spectrum antibiotic developed and marketed by The Medicines, offering superior antibacterial spectrum and clinical efficacy compared to vancomycin. It was approved by the FDA on August 6, 2014. The approved compound is oritavancin diphosphate, marketed under the brand name "Orbactiv®," and is approved by the FDA for the treatment of adult patients with abscesses and septicemia (ABSSSI) caused by susceptible Gram-positive bacteria, including MRSA.

[0005] Orivacin is a novel semi-synthetic lipoglycopeptide antibiotic that exhibits good activity against multidrug-resistant (MDR) Staphylococcus, Enterococcus, and Streptococcus. Its long half-life allows it to be used in clinical practice as a single-dose treatment regimen for the entire course of treatment. This is the first and only single-dose antibiotic approved by the FDA for the treatment of ABSSSI.

[0006] A82846B is a key intermediate in the chemical synthesis of Orivacin, and its only current source is microbial fermentation. This compound was initially discovered by Eli Lilly & Company in the 1990s from *Nocardia orientalis* (…). Nocardia orientalisThe fermentation broth of certain strains has been found to contain A82846B. US Patent 5312738 (filed May 9, 1990) describes the producing strain, fermentation, and extraction process of A82846B. However, the fermentation process reported in this patent and subsequent literature has the following three defects: First, the fermentation potency of the target component A82846B is low (150 mg / L); second, the fermentation medium uses large amounts of expensive raw materials such as casein and tyrosine, resulting in high fermentation costs; third, the fermentation broth contains three homologues simultaneously: A82846A, A82846B, and A82846C (referred to as "component A," "component B," and "component C," respectively). Among them, component A, as an impurity, accounts for an excessively high proportion (A / B ratio of 3-4), thus causing difficulties in separating the target product, low extraction yield, and high cost. These defects have hindered the industrial development of this novel antibiotic. By selecting and breeding production strains and developing supporting fermentation processes, the use of high-priced raw materials in the fermentation medium can be reduced, further improving the fermentation potency, especially by increasing the proportion of component B in the fermentation broth (i.e., reducing the A / B ratio). This will have great application value for the industrialization development of this variety.

[0007] The molecular formula of Orivancin intermediate A82846B is C 73 H 88 Cl2N 10 O 26 CAS No.: 118395-73-6; Structural formula is shown in Formula 1:

[0008]

[0009] Formula 1.

[0010] The main impurity component A has the research and development code A82846A and the molecular formula C. 73 H 89 ClN 10 O 26 CAS No.: 110865-90-2; Structural formula is shown in Formula 2:

[0011]

[0012] Formula 2

[0013] In nature, the genetic material of microorganisms spontaneously undergoes mutations during reproduction, resulting in new traits. However, the probability of natural mutation is very low, only 10%. -9 ~10 -6This approach cannot meet the needs of scientific research and practical production. Artificial mutagenesis can significantly increase the mutation rate of strains and accelerate the breeding process. UV mutagenesis is a classic mutagenesis technique widely used in the field of microbial mutagenesis breeding, offering advantages such as ease of operation, stable results, and safety. Its working principle is as follows: irradiating microbial cells with short-wave ultraviolet light (λ=254 nm) in the UVC band causes damage to the DNA molecules of the tested cells (breakage or formation of pyrimidine dimers), and further generates mismatches and other mutations during the subsequent repair process, thereby forming new traits in daughter cells. The DNA damage caused by UV mutagenesis and the resulting mutations are random; therefore, it is necessary to screen a large number of samples to identify the desired target mutant strains. Summary of the Invention

[0014] The purpose of this invention is to provide a strain that produces the intermediate A82846B of orivanthan, the strain having the accession number CGMCC No.17931.

[0015] The structural formula of the intermediate A82846B of Origen is shown in Formula 1:

[0016]

[0017] Formula 1.

[0018] Preservation information of the biological material of this strain:

[0019] This strain was deposited for patent preservation at the China General Microbiological Culture Collection Center (CGMCC) on June 14, 2019. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Postcode 100101); the accession number is CGMCC No. 17931; the biological material (strain) is JH100-32B16B; and the classification name is *Pseudomonas desertis*. Kibdelosporangium aridum .

[0020] Nocardia orientalis ( Nocardia orientalis The natural isolate JH100, which produces compounds of the A82846 series, was used as the original starting strain. After the first round of mutagenesis and re-screening, the mutant strain JH100-32B was obtained. Then, the mutant strain JH100-32B was used as the subsequent starting strain for multiple rounds of ultraviolet mutagenesis. A mutant strain with unique production performance that can be fermented in a low-nutrient medium without readily available organic nitrogen sources was obtained and named JH100-32B16B.

[0021] This strain possesses the following biological characteristics:

[0022] (1) Morphological characteristics

[0023] The strain can differentiate or not differentiate aerial hyphae on different solid culture media; the aerial hyphae are generally white. Substrate hyphae are pale yellow or brown. Colonies are raised, forming the characteristic ring-shaped morphology of this fungus. In the later stages of cultivation, white conidia are generated at the top of the colonies. On ISP-2 solid medium, substrate hyphae can produce a pale brown water-soluble pigment; no soluble pigment has been observed on other solid culture media formulations.

[0024] The strain forms mycelial balls in a liquid culture environment, and the viscosity of the culture medium gradually increases.

[0025] The strain is morphologically described under a microscope as a filamentous, multicellular, prokaryotic, Gram-positive fungus. The hyphae are approximately 0.5 μm in diameter.

[0026] (2) Cultivation characteristics

[0027] The strain can grow well in both solid and liquid culture media at 20-35℃ and pH 4.5-7.8.

[0028] (3) Physiological and biochemical characteristics

[0029] Regarding carbon source utilization, the strain can utilize most carbon sources except lactose, sorbitol, erythritol, and raffinose; regarding nitrogen sources, the strain cannot utilize lysine and reduced nitrates; the strain can peptone milk but cannot liquefy gelatin.

[0030] This invention overcomes the problems of high fermentation costs caused by the presence of high-priced raw materials such as tyrosine and hydrolyzed casein in the fermentation medium for producing the intermediate A82846B of Orivancin using existing strains, as well as the low fermentation potency of the target product; and the high impurity-to-product ratio (A / B value) in the fermentation broth, resulting in low product extraction yield and high costs.

[0031] Another object of the present invention is to provide a method for preparing Orivastar intermediate A82846B by fermentation.

[0032] The strain with preservation number CGMCC No.17931 was fermented in a low-nutrient medium without readily available organic nitrogen sources, and the intermediate A82846B of Orivancin was obtained from the fermentation broth.

[0033] The oligo-enriched culture medium comprises the following components (g / L): 0-30 g of readily available carbon source, 10-60 g of slowly available carbon source, and 10-50 g of slowly available nitrogen source. The readily available carbon source is one or more of glucose, sucrose, and maltose; the slowly available carbon source is one or more of maltodextrin and corn starch; and the slowly available nitrogen source is one or more of soybean meal, cottonseed meal, and peanut meal. The oligo-enriched culture medium does not contain readily available organic nitrogen sources. The pH value of the oligo-enriched culture medium is the natural pH value presented after the components are prepared in the specified proportions.

[0034] Preferably, the oligoculture medium contains the following components (g / L): glucose 10-30, maltodextrin 20-60, molasses 10-30, cottonseed meal 10-50, and natural pH.

[0035] More preferably, the content of each component of the depleted culture medium is (g / L): glucose 20-30, maltodextrin 30-50, molasses 15-25, cottonseed meal 10-20, pH 6.0-7.5.

[0036] The fermentation temperature is 20~35℃, preferably 25~30℃; the fermentation cycle is 240~500 hours, preferably 288~360 hours.

[0037] When the fermentation is carried out in a 10 L fermenter, the process parameters are as follows: initial liquid volume is 4~7 L, preferably 4.5~6.0 L; stirring speed is 200~1000 rpm, preferably 300~800 rpm; aeration rate is 0.3~1.0 vvm, preferably 0.5~1.0 vvm; dissolved oxygen level in fermentation broth is 0~100%, preferably 20~50%; fermentation inoculum size is 5~15%, preferably 5~10%.

[0038] When the fermentation is carried out in a 30 L fermenter, the process parameters are as follows: initial liquid volume is 10-20 L, preferably 12-15 L; stirring speed is 200-1000 rpm, preferably 200-700 rpm; aeration rate is 0.2-1.0 vvm, preferably 0.3-0.5 vvm; dissolved oxygen level in fermentation broth is 0-100%, preferably 20-30%; and inoculum size is 5-15%, preferably 5-8%.

[0039] The fermentation process, when carried out in a fermentation tank, also includes a feeding operation.

[0040] The feed contains the following components: 0-50% organic carbon source and 0-30% organic nitrogen source. The organic carbon source is one or more of sucrose, maltose, molasses, and maltodextrin; the organic nitrogen source is one or more of peptone, corn steep liquor, fish meal, and soy protein powder.

[0041] Preferably, the composition of the feed is: molasses 10-30%, sucrose 0-50%, and peptone 0-30%.

[0042] The feeding operation described above involves adding the aforementioned feed material into the main fermenter at a certain flow rate. The feeding rate is determined based on a comprehensive evaluation of indicators such as dissolved oxygen, reducing sugar, and amino nitrogen during the fermentation process.

[0043] The following preparation method is a summary and refinement based on the examples. Please check if it is accurate:

[0044] According to one embodiment, the preparation method of Orivancin intermediate A82846B includes the following steps:

[0045] The JH100-32B16B strain preserved in glycerol tubes was transferred to ISP-2 slant culture and activated in a 28℃ constant temperature and humidity incubator for 5-7 days. The activated bacterial growth was then transferred to seed bottles. The seed bottle volume was 30 mL of seed culture medium in a 250 mL Erlenmeyer flask. The seed culture medium formula (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ with shaking for 48 h at 200 rpm. The mature seed culture was then transferred to fermentation shake flasks for shake-flask fermentation. The shake-flask fermentation medium formula (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The fermentation bottle volume was 20 mL of culture medium in a 250 mL Erlenmeyer flask. The inoculum size for the fermentation shake flasks was 10%. The fermentation conditions were 28℃, constant temperature shaker speed of 200 rpm, and shaking culture for 7 days.

[0046] According to one embodiment, the preparation method of Orivancin intermediate A82846B includes the following steps:

[0047] The JH100-32B16B strain preserved in glycerol tubes was transferred to ISP-2 slant culture and activated in a 28℃ constant temperature and humidity incubator for 5-7 days. The activated bacterial growth was then transferred to seed bottles. Each seed bottle contained 125 mL of seed culture medium in a 500 mL Erlenmeyer flask. The seed culture medium formula (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ for 48 h with shaking at 200 rpm. The mature seed culture was then transferred to a fermenter for on-tank fermentation. The fermenter substrate formula (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The initial volume of the fermentation medium was 6 L. After adding the components of the fermentation medium, deionized water was added to dissolve them. The pH was adjusted to 7.5 with 5N NaOH solution. The medium was sterilized at 121℃ for 30 min and then cooled to room temperature before inoculation. The inoculation amount in the fermenter was 8-10%. The fermentation temperature was 28℃, the initial stirring speed was 300 rpm, and the initial aeration rate was 0.5 vvm. Subsequently, the stirring speed and aeration rate were gradually increased according to the dissolved oxygen situation. Feeding was started after 48 h of fermentation. The feed formula was: molasses 30%, sucrose 10%, and peptone 20%. The feeding method was constant flow at a rate of 100 mL / d. After 384 h of main fermentation, the fermenter was discharged, and the concentration of the target product A82846B was determined by HPLC.

[0048] According to one embodiment, the preparation method of Orivancin intermediate A82846B includes the following steps:

[0049] The JH100-32B16B strain preserved in glycerol tubes was transferred to ISP-2 slant culture and activated in a 28℃ constant temperature and humidity incubator for 5-7 days. The activated bacterial growth was then transferred to seed bottles. Each seed bottle contained 250 mL of seed culture medium in a 1 L Erlenmeyer flask. The seed culture medium formula (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ for 48 h with shaking at 200 rpm. The mature seed culture was then transferred to a fermenter for on-tank fermentation. The fermenter substrate formula (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The initial volume of the primary fermenter was 16 L. After adding the components of the fermentation medium, deionized water was added to dissolve them. The pH was adjusted to 7.5 with 5N NaOH solution. The culture was sterilized at 121℃ for 30 min and then cooled to room temperature for inoculation. The inoculation volume was 1 L of seed culture. The fermentation temperature was 28℃, the initial stirring speed was 300 rpm, and the initial aeration rate was 0.5 vvm. The stirring speed and aeration rate were then gradually increased based on dissolved oxygen levels. Feeding was initiated after 48 h of fermentation. The feed composition was: 30% molasses, 10% sucrose, and 20% peptone. The feeding method was DO-dependent, maintaining DO between 10% and 30%. After 312 h of primary fermentation, the fermenter was discharged, and the concentration of the target product A82846B was determined by HPLC.

[0050] The present invention also provides a seed preparation process for the fermentation production of Orivanstar intermediate A82846B.

[0051] The seed preparation process includes activating strain CGMCC No.17931, transferring it to a shake flask, and then transferring the mature shake flask seed to a tank to finally obtain the seed culture medium required for the main fermentation.

[0052] The aforementioned strain activation refers to the process of transferring strain CGMCC No. 17931 onto an activation medium for cultivation. The strain activation culture uses common actinomycete media, preferably ISP-2 solid medium. The medium is in the form of plates or slant agar. The activation culture conditions are: temperature 15–35℃, preferably 25–30℃; culture period 3–10 days, preferably 5–7 days.

[0053] The preparation of the shake flask culture refers to the process of transferring the activated desert spirocystis CGMCC No.17931 culture to a seed culture medium and culturing it in a shake flask.

[0054] The seed culture medium contains the following components (g / L): glucose 5-20, maltodextrin 10-30, hydrolyzed casein 3-10, yeast extract 3-10, and natural pH.

[0055] Preferably, the components of the shake flask seed culture medium are (g / L): glucose 8-15, maltodextrin 15-20, hydrolyzed casein 5-10, yeast extract 5-10, pH 6.5-7.5.

[0056] The culture of the shake flask seed culture is carried out in a shake flask. Preferably, the shake flask has a capacity of 1 L and contains 250 mL of shake flask seed culture medium; the shake flask seed culture medium is sterilized using conventional autoclaving in the art; the sterilized shake flask seed culture medium is inoculated with the aforementioned activated culture of *Pseudomonas aeruginosa* CGMCC No. 17931, with an inoculation amount preferably of 2-8%; the inoculated shake flask is then cultured on a constant temperature shaker, with a shaker speed preferably of 150-250 rpm, a temperature preferably of 25-30°C, and a culture period preferably of 1-2 days.

[0057] The preparation of the seed in the seed tank refers to the process of transferring the shake flask inoculum of *Desert Species* CGMCC No. 17931 into a seed culture medium and culturing it in a seed tank.

[0058] The seed culture was carried out in a 10 L seed tank. The process parameters were as follows: initial liquid volume in the seed tank was 4-7 L, preferably 5-6 L; stirring speed was 100-600 rpm, preferably 200-400 rpm; aeration rate was 0.1-0.5 vvm, preferably 0.3-0.5 vvm; dissolved oxygen level in the fermentation broth was 0-100%, preferably 20-30%; initial inoculum size was 2-10%, preferably 3-5%. The culture time was determined based on the bacterial density, pH value, and DO trend, generally 24-48 h.

[0059] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0060] The reagents and raw materials used in this invention are all commercially available.

[0061] The positive and progressive effects of this invention are as follows:

[0062] This invention provides a strain CGMCC No. 17931, a producer of orivacin intermediate (A82846B), obtained through UV mutagenesis. This strain exhibits stable genetic traits and can produce orivacin intermediate A82846B using a low-nitrogen medium (without readily available nitrogen sources) as the primary fermentation medium. Furthermore, the impurity-to-product ratio (A / B value) in the fermentation broth is significantly lower than that of the control. Using this strain, along with the fermentation medium and control process developed based on it, an A82846B fermentation titer of 1450 mg / L can be achieved in a 30 L fermenter, reducing the raw material cost by more than 90% compared to the control process. Moreover, the impurity-to-product ratio (A / B value) in the fermentation broth is only 1.5–2.0 (compared to 3–5 in previous literature reports), greatly improving the yield of subsequent extraction steps and contributing to further cost reduction.

[0063] The desert spirocyst strain selected in this invention does not require readily available nitrogen sources and expensive raw materials such as casein. It can produce A82846B component by fermentation using low-cost, low-nutrient culture medium. The fermentation titer is high, and the proportion of B component is much higher than that of the control process, which significantly reduces fermentation and extraction costs and is beneficial to the industrial production of this variety.

[0064] This invention utilizes UV irradiation to induce multiple rounds of artificial mutagenesis in the starting strain, resulting in a *Streptomyces desertica* mutant strain with excellent production performance. Based on this mutant strain, targeted optimization of culture medium components and development of fermentation processes were conducted, improving fermentation potency, reducing the impurity-to-product ratio (to half that of the control strain), significantly lowering fermentation costs (to 1 / 10 of the control level), and simplifying the extraction process. Attached Figure Description

[0065] Figure 1 This is a pedigree chart of strain JH100-32B16B (CGMCC No. 17931).

[0066] Figure 2 This is the HPLC analysis spectrum of the fermentation broth from Example 3.

[0067] Figure 3 This is the HPLC analysis spectrum of the fermentation broth from Example 7.

[0068] Figure 4 This is the LC-MS spectrum of A82846B produced in the fermentation broth of Example 7.

[0069] Figure 5 This is the A82846B potency curve during the fermentation process of Example 7. Detailed Implementation

[0070] The invention will be further illustrated below with reference to specific examples and accompanying drawings. It should be understood that these examples are only for better illustrating the invention and are not intended to limit the invention to the technical parameters of the described embodiments.

[0071] Example 1

[0072] Obtaining strain JH100-32B16B (CGMCC No.17931).

[0073] Strain JH100 was obtained by natural isolation from a Nocardia orientalis strain producing the A82846 series of compounds (Hamill et al. A82846ANTIBIOTICS [P]. USP 5312738, May 17, 1994). Subsequent mutagenesis breeding operations were initiated using JH100 as the starting strain.

[0074] Cell preparation: First, serially dilute the JH100 liquid culture with sterile water. Add glass beads to the dilution tubes and shake thoroughly on a shaker to ensure the hyphae are dispersed. Use a sterile graduated pipette to draw approximately 0.1 mL of the appropriately diluted bacterial suspension and add it to a sterile ISP2 plate. Spread the plate using an L-shaped glass scraper. Prepare several spread plates in this manner.

[0075] First round of mutagenesis: The starting strain (JH100) described above was spread on plates and irradiated under a UV lamp. The UV irradiation parameters were: UV wavelength 254 nm, lamp power 8 W, irradiation spacing of test samples 20 cm, and irradiation times of 30 s, 1 min, 2 min, 5 min, and 10 min. The control group (CK) was not irradiated. All operations except UV irradiation were performed in a dark environment. All irradiated test and control plates were incubated in a 28℃ incubator for 5 days.

[0076] Initial screening: The plates from the test group and control group described above were removed from the incubator, and colony growth was observed. The control group showed normal growth, indicating no abnormalities in culture conditions or bacterial strains, and the experimental results were reliable. The lethality rates of the 2-min, 5-min, and 10-min irradiation groups were all >99%. Several single colonies were randomly picked from these three groups of plates, transferred to slant agar, numbered, and incubated at 28℃ for 48 h. The formulation used for the slant agar was still ISP-2. Bacterial growth was picked from the cultured slant agar and transferred to inoculation bottles. Glycerol tubes were also prepared and stored for subsequent rescreening.

[0077] The seed bottle formulation is as follows (g / L): glucose 15, maltodextrin 15, hydrolyzed casein 10, yeast extract 10, pH 7.2. The seed bottle contains 30 mL of culture medium in a 250 mL Erlenmeyer flask. The seed bottle culture conditions are 28°C, constant temperature shaker at 180 rpm, for 48 h. The glycerol tubes are stored at -80°C in a freezer.

[0078] After seed culture, the culture was transferred to a fermentation flask for shake-flask fermentation. The fermentation medium composition (g / L) was: glucose 30, maltodextrin 30, molasses 25, hydrolyzed casein 10, yeast extract 10, cottonseed meal 10, pH 7.5. Each 250 mL Erlenmeyer flask contained 20 mL of the medium. The seed culture inoculum was 10%. Fermentation conditions were 28℃, constant temperature shaker speed 200 rpm, and shaking culture for 10 days. After fermentation, the concentration of the target product A82846B was detected by HPLC, and the A / B ratio was calculated. These two indicators were used as evaluation criteria for the strain's performance.

[0079] Secondary screening: From the results of the initial screening, mutant strains with high fermentation titer and low A / B ratio (A82846B) were selected for secondary seed preparation and shake-flask fermentation. The seed preparation, fermentation medium, and conditions were the same as in the initial screening, and their fermentation titer and A / B ratio were examined. Mutants showing better performance in both indicators than the control group (starting strain), good data reproducibility between the two screenings, and stable genetic traits were selected for a second round of mutagenesis screening. This round of mutagenesis yielded a superior mutant strain, JH100-32B, with an A82846B shake-flask fermentation titer >400 mg / L and an A / B ratio of approximately 3.

[0080] The second round and subsequent mutagenesis: The superior mutant strain JH100-32B obtained from the first round of mutagenesis was used as the starting strain for subsequent operations, and UV mutagenesis was performed again using the same method as the first round of mutagenesis described above. The initial and secondary screening operations after mutagenesis were the same as in the first round of mutagenesis. The relatively optimal strain was selected from the subsequent mutagenesis for further mutagenesis. A total of three rounds of subsequent mutagenesis were performed, resulting in a mutant strain numbered JH100-32B16B. This strain can perform shake-flask fermentation using a low-nitrogen medium lacking readily available organic nitrogen sources. In a 250 mL shake flask, the fermentation titer of A82846B was >400 mg / L, and the A / B ratio was 1.2–1.5. The low-nitrogen medium formula was (g / L): glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5.

[0081] The mutant strain JH100-32B16B was submitted to the China General Microbiological Culture Collection Center (CGMCC) for patent preservation on June 14, 2019, with registration number CGMCC No. 17931. The biological material (strain) is JH100-32B16B; its classification name is *Pseudomonas desertis*. Kibdelosporangium aridum .

[0082] Example 2

[0083] Identification of strain JH100-32B16B (CGMCC No.17931).

[0084] (1) Morphological characteristics of strain JH100-32B16B

[0085] After culturing strain JH100-32B16B on solid culture media (plates) with different formulations at 28℃ for 5-7 days, the colony morphology was observed and shown in Table 1.

[0086] Table 1

[0087] Czapek's medium, glucose asparagine medium, glycerol asparagine medium, inorganic salt starch medium, ISP-2 medium, oat flour medium, Gao's No. 1 medium, Santay's medium White, white, white, no, no, little; white, no, brown. Cork yellow, creamy yellow, light milky white, coffee brown, light cork yellow, light milky white, coffee brown None None None Light coffee brown None None None

[0088] Aerial hyphae of strain JH100-32B16B were selected and observed under an optical microscope. Their characteristics were: straight, flexible, hook-shaped, loose and tight spiral filaments; columnar spores with sporangial structures.

[0089] (2) Culture and physiological and biochemical characteristics of strain JH100-32B16B

[0090] The physiological and biochemical characteristics of strain JH100-32B16B during the culture process were investigated. Table 2 shows the carbon source utilization characteristics; Table 3 shows the nitrogen source utilization characteristics of the strain; and Table 4 shows other physiological and biochemical characteristics of the strain.

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] As shown in Table 3, this strain can utilize all common amino acids except for lysine.

[0096] Table 4

[0097]

[0098] As shown in Table 4, this strain does not produce nitrate reductase and cannot liquefy gelatin; however, it is positive for milk peptone reaction and can produce tyrosinase and amylase.

[0099] (3) 16S rRNA sequence determination of strain JH100-32B16B

[0100] The 16S rRNA gene of strain JH100-32B16B was sequenced, and the sequence is shown in the attached sequence listing. This sequence was then subjected to BLAST alignment analysis in GenBank, and the results of the first six sequences sorted by homology are shown in Table 5.

[0101] Table 5

[0102]

[0103] In summary, based on the comprehensive analysis of experimental data including the culture characteristics, cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence of the strain, and referring to Bergey's Manual of Systematic Bacteriology and relevant research papers in the International Journal of Systematic and Evolutionary Microbiology, JH100-32B16B is identified as *Pseudomonas desertis*. Kibdelosporangium aridum .

[0104] Example 3

[0105] Shake-flask fermentation of control strain JH100. JH100 culture, preserved in glycerol tubes, was transferred to ISP-2 slant agar and activated in a 28℃ constant temperature and humidity incubator for 5–7 days. Activated bacterial growth was collected and transferred to seed bottles. The seed bottle composition was: 30 mL seed culture medium in a 250 mL Erlenmeyer flask. The seed culture medium formulation (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottle was incubated on a shaker at 28℃ for 48 h at 200 rpm. Mature seed culture was then transferred to fermentation shake flasks for shake-flask fermentation. The shake-flask fermentation medium formulation (g / L) was: glucose 30, maltodextrin 30, molasses 25, hydrolyzed casein 10, yeast extract 10, cottonseed meal 10, pH 7.5. The fermentation bottle composition was: 20 mL culture medium in a 250 mL Erlenmeyer flask. The inoculum size in the fermentation flasks was 10%. Fermentation conditions were 28℃, constant temperature shaker speed 200 rpm, and shaking culture for 7 days. After fermentation, the concentration of the target product A82846B was determined by HPLC, and the A / B ratio was calculated. The product detection conditions were as follows: the pH of the fermentation broth was adjusted to 10.5 with 5N sodium hydroxide solution, centrifuged, and the supernatant was filtered through a 0.22 μm microfiltration membrane before HPLC analysis. The HPLC system used was a Shimadzu LC-2030 series high-performance liquid chromatograph, with an Agilent ZORBAX Eclipse plus C18 column (4.6×250 mm, 5.0 μm) and a mobile phase of water:acetonitrile. The titer of A82846B in the 7-day shake-flask fermentation broth of the control strain JH100 was 198 mg / L, with an A / B ratio of 2.2.

[0106] Example 4

[0107] Shake-flask fermentation of strain JH100-32B16B. The JH100-32B16B strain, preserved in glycerol tubes, was transferred to ISP-2 agar slants and activated in a 28℃ constant temperature and humidity incubator for 5–7 days. The activated bacterial growth was then transferred to seed bottles. The seed bottle volume was 30 mL of seed culture medium in a 250 mL Erlenmeyer flask. The seed culture medium formula (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ for 48 h at 200 rpm. The mature seed culture was then transferred to fermentation shake flasks for shake-flask fermentation. The shake-flask fermentation medium formula (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The fermentation bottle volume was 20 mL of culture medium in a 250 mL Erlenmeyer flask. The inoculum size in the fermentation shake flasks was 10%. Fermentation conditions were 28℃, constant temperature shaker speed 200 rpm, and shaking culture for 7 days. After fermentation, the concentration of the target product A82846B was determined by HPLC, and the A / B ratio was calculated. The product detection conditions were as follows: the pH of the fermentation broth was adjusted to 10.5 with 5N sodium hydroxide solution, centrifuged, and the supernatant was filtered through a 0.22 μm microfiltration membrane before HPLC analysis. The HPLC system used was a Shimadzu LC-2030 series high-performance liquid chromatograph, with an Agilent ZORBAX Eclipse plus C18 column (4.6 × 250 mm, 5.0 μm) and a mobile phase of water:acetonitrile. The titer of A82846B in the 7-day shake flask fermentation broth of the control strain JH100-32B16B was 531 mg / L, with an A / B ratio of 1.6.

[0108] Example 5

[0109] Fermentation of control strain JH100 in a 10L fermenter. JH100 culture preserved in glycerol tubes was transferred to ISP-2 slant culture and activated in a 28℃ constant temperature and humidity incubator for 5–7 days. Activated bacterial growth was then transferred to seed bottles. Each seed bottle contained 125 mL of seed culture medium in a 500 mL Erlenmeyer flask. The seed culture medium formulation (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ with shaking for 48 h at 200 rpm. Mature seed culture was then transferred to the fermenter for on-tank fermentation. The fermenter substrate formulation (g / L) was: glucose 30, maltodextrin 30, molasses 25, hydrolyzed casein 10, yeast extract 10, cottonseed meal 10, pH 7.5. The initial volume of the fermentation medium was 6 L. After adding the components of the fermentation medium, deionized water was added to dissolve them. The pH was adjusted to 7.5 with 5N NaOH solution. The medium was sterilized at 121℃ for 30 min and then cooled to room temperature before inoculation. The inoculation amount in the fermenter was 8-10%. The fermentation temperature on the tank was 28℃, the initial stirring speed was 300 rpm, and the initial aeration rate was 0.5 vvm. Subsequently, the stirring speed and aeration rate were gradually increased according to the dissolved oxygen situation. Feeding was started after 48 h of fermentation. The feed formula was 50% molasses and 10% hydrolyzed casein. The feeding method was DO-coupled feeding, with the feed pump operation coupled to the DO value, maintaining the DO level in the range of 10-30%. After 213 h of main fermentation, the tank was discharged, and the concentration of the target product A82846B was detected by HPLC, and the A / B ratio was calculated. The product detection conditions were as follows: the pH of the fermentation broth was adjusted to 10.5 with 5N sodium hydroxide solution, centrifuged, and the supernatant was filtered through a 0.22 μm microfiltration membrane before HPLC detection. The HPLC system used was a Shimadzu LC-2030 series high-performance liquid chromatograph with an Agilent ZORBAX Eclipse plus C18 column (4.6 × 250 mm, 5.0 μm) and a water:acetonitrile mobile phase. The control strain JH100 was fermented in a 10L fermenter for 213 h, and the titer of A82846B in the fermentation broth was 544 mg / L, with an A / B ratio of 3.1.

[0110] Example 6

[0111] Fermentation of strain JH100-32B16B in a 10L fermenter. The JH100-32B16B strain, preserved in glycerol tubes, was transferred to ISP-2 agar slants and activated in a 28℃ constant temperature and humidity incubator for 5–7 days. Activated bacterial colonies were then transferred to seed bottles. Each seed bottle contained 125 mL of seed culture medium in a 500 mL Erlenmeyer flask. The seed culture medium formulation (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ with shaking for 48 h at 200 rpm. The mature seed culture was then transferred to the fermenter for on-tank fermentation. The fermenter substrate formulation (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The initial volume of the fermentation medium was 6 L. After adding the components of the fermentation medium, deionized water was added to dissolve them. The pH was adjusted to 7.5 with 5N NaOH solution. The medium was sterilized at 121℃ for 30 min and then cooled to room temperature before inoculation. The inoculation amount of the fermenter was 8-10%. The fermentation temperature was 28℃, the initial stirring speed was 300 rpm, and the initial aeration rate was 0.5 vvm. Subsequently, the stirring speed and aeration rate were gradually increased according to the dissolved oxygen situation. Feeding was started after 48 h of fermentation. The feed formula was: molasses 30%, sucrose 10%, and peptone 20%. The feeding method was constant flow at a rate of 100 mL / d. After 384 h of main fermentation, the fermenter was discharged, and the concentration of the target product A82846B was detected by HPLC, and the A / B ratio was calculated. The product detection conditions were as follows: the fermentation broth was adjusted to pH 10.5 with 5N sodium hydroxide solution, centrifuged, and the supernatant was filtered through a 0.22 μm microfiltration membrane before HPLC detection. The HPLC system used was a Shimadzu LC-2030 series high-performance liquid chromatograph with an Agilent ZORBAX Eclipse plus C18 column (4.6 × 250 mm, 5.0 μm) and a water:acetonitrile mobile phase. Strain JH100-32B16B was fermented in a 10L fermenter for 384 h. The titer of A82846B in the fermentation broth was 1450 mg / L, and the A / B ratio was 1.86.

[0112] Example 7

[0113] Fermentation of strain JH100-32B16B in a 30L fermenter. The JH100-32B16B strain, preserved in glycerol tubes, was transferred to ISP-2 agar slants and activated in a 28℃ constant temperature and humidity incubator for 5–7 days. The activated bacterial growth was then transferred to seed bottles. Each seed bottle contained 250 mL of seed culture medium in a 1L Erlenmeyer flask. The seed culture medium formulation (g / L) was: glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.2. The seed bottles were incubated on a shaker at 28℃ with shaking for 48 h at 200 rpm. The mature seed culture was then transferred to the fermenter for on-tank fermentation. The fermenter substrate formulation (g / L) was: glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.5. The initial volume of the primary fermenter was 16L. After adding the components of the fermentation medium, deionized water was added to dissolve them. The pH was adjusted to 7.5 with 5N NaOH solution. The culture was sterilized at 121℃ for 30 min and then cooled to room temperature for inoculation. The inoculation volume was 1 L of seed culture. The fermentation temperature was 28℃, the initial stirring speed was 300 rpm, and the initial aeration rate was 0.5 vvm. The stirring speed and aeration rate were then gradually increased based on dissolved oxygen levels. Feeding was initiated after 48 h of fermentation. The feed composition was: 30% molasses, 10% sucrose, and 20% peptone. The feeding method was DO-dependent, maintaining DO between 10% and 30%. After 312 h of primary fermentation, the fermenter was discharged, and the concentration of the target product A82846B was determined by HPLC, and the A / B ratio was calculated. The product detection conditions were as follows: the pH of the fermentation broth was adjusted to 10.5 with 5N sodium hydroxide solution, centrifuged, and the supernatant was filtered through a 0.22 μm microfiltration membrane before HPLC detection. The HPLC system used was a Shimadzu LC-2030 series high-performance liquid chromatograph with an Agilent ZORBAX Eclipse plus C18 column (4.6 × 250 mm, 5.0 μm) and a water:acetonitrile mobile phase. Strain JH100-32B16B was fermented in a 10L fermenter for 312 h. The titer of A82846B in the fermentation broth was 1363 mg / L, and the A / B ratio was 1.43.

[0114] Those skilled in the art can recombine and modify the operation procedures and technical parameters listed in the above embodiments in accordance with common sense in the field. These modifications are equivalent to natural extensions of the embodiments of the present invention and still fall within the scope of the claims of the present invention. sequence list <110> Shanghai Jianhe Pharmaceutical Technology Co., Ltd. <120> An intermediate-producing bacterium of Orivancin and its application <141> 2020-04-04 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1390 <212> DNA <213> Kibdelosporangium aridum <400> 1 catgcagtcg agcggtaagg cctttcgggg tacacgagcg gcgaacgggt gagtaacacg 60 tgggtaatct gccctgcact ctgggataag cccggggaaac tgggtctaat accggatacg 120 actcgcgcac gcatctgtgt gggtggaaag ttccggcggt gcaggatgag cccgcggcct 180 atcagcttgt tggtggggtg atggcctacc aaggcgacga cgggtagccg gcctgagagg 240 gcgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300 aatattgc aatgggcgaa agcctgacgc agcgacgccg cgtgagggat gacggccttc 360 gggttgtaaa cctctttcgc cagggacgaa gcgagagtga cggtacctgg agaagaagca 420 ccggctgact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt gtccggaatt 480 attgggcgta aagagctcgt aggcggtttg ttgcgtcggt cgtgaaaacc tggggcttaa 540 ccctgggctt gcggtcgata cgggcagact tgagttcggt aggggagact ggaattcctg 600 gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc gggtctctgg 660 gccgatactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720 gtccacgccg taaacgttgg gcgctaggtg tggggctctt tccacgggtt ccgtgccgta 780 gctaacgcat taagcgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840 tgacgggggc ccgcacaagc ggcggagcat gtggattaat tcgatgcaac gcgaagaacc 900 ttacctgggc ttgacatgcg ccagacatcc ctagagatag ggcttccctt gtggttggtg 960 tgcaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020 cgagcgcaac cctcgttcca tgttgccagc acgttatggc ggggactcat gggagactgc 1080 cggggtcaac tcggaggaag gtggggatga cgtcaagtca tcatgcccct tatgtccagg 1140 gcttcacaca tgctacaatg gctagtacag agggctgcga gaccgtgagg tggagcgaat 1200 cccttaaagc tggtctcagt tcggattggg gtctgcaact cgaccccatg aagtcggagt 1260 cgctagtaat cgcagatcag caacgctgcg gtgaatacgt tcccgggcct tgtacacacc 1320 gcccgtcacg tcacgaaagt cggtaacacc cgaagcccac ggcctaaccc cttgtgggag 1380 ggagtgtcga 1390

Claims

1. A method for preparing Orivans intermediate A82846B, wherein, The structural formula of Orivancin intermediate A82846B is shown in Formula 1: Formula 1; The preparation method includes the following steps: fermenting strain CGMCC No.17931 in a low-nutrient medium without readily available organic nitrogen sources to obtain the Orivancin intermediate A82846B from the fermentation broth; the steps are as follows: The strain with accession number CGMCC No.17931, preserved in glycerol tubes, was transferred to ISP-2 slant and activated in a constant temperature and humidity incubator at 28℃ for 5–7 days; the activated bacterial growth was then picked and transferred to a seed bottle. The seed culture medium, consisting of 250 mL of seed culture medium in a 1 L Erlenmeyer flask, was formulated as follows (g / L): glucose 15, maltodextrin 15, hydrolyzed casein 3, yeast extract 3, pH 7.

2. The seed culture was incubated on a shaker at 28℃ for 48 h at 200 rpm. Mature seeds were then transferred to a fermenter for onboard fermentation. The fermentation medium was formulated as follows (g / L): glucose 30, maltodextrin 30, molasses 25, cottonseed meal 10, pH 7.

5. The initial volume of the main fermenter was 16 L. After adding the fermentation medium components, deionized water was added to dissolve them. The pH was adjusted to 7.5 with NaOH solution, sterilized at 121℃ for 30 minutes, and then cooled to room temperature for inoculation. The inoculation volume was 1L of seed culture, the fermentation temperature was 28℃, the initial stirring speed was 300rpm, and the initial aeration rate was 0.5vvm. Subsequently, the stirring speed and aeration rate were gradually increased according to the dissolved oxygen situation. Feeding was started after 48 hours of fermentation. The feed formula was: molasses 30%, sucrose 10%, and peptone 20%. The feeding method was DO-dependent, maintaining DO in the range of 10-30%. After 312 hours of main fermentation, the product was discharged from the tank, and the concentration of the target product A82846B was detected by HPLC.

Images