DD233753A3 - PROCESS FOR PRODUCING NOURSEOTHRICIN - Google Patents

Abstract

The invention relates to the production of the streptothricin antibiotic nouresothricin by microbial fermentations by influencing the phosphate metabolism. For this purpose, two manipulations are carried out consecutively or individually: Lowering the initial concentration of soluble and available phosphate to a low level during the sterilization of the nutrient medium recharge of phosphate by addition of phosphate-containing substances after a suitable addition regime. Lowering the initial concentration of Available phosphate is obtained by increasing the p H values during sterilization and by precipitation of phosphate-containing sediments in the presence of phosphate-precipitating ions. The acidity of the fermentation media is either set directly to alkaline p H values during sterilization or indirectly increased by the presence of higher sulfate concentrations in addition to calcium carbonate during direct steam sterilization. Phosphate supply of the phosphate-containing substances takes place after an optimal regime during the fermentation while avoiding further limitations or other unfavorable conditions.

Description

Field of application of the invention

The invention relates to the production of the streptothricin antibiotic nourseothricin by the targeted influencing of the microbial fermentation. It is important for the microbial industry.

Characteristic of the known technical solution

Three major repressive influences by the substrates are mentioned in the literature in the context of secondary metabolite biosynthesis, the

1. by glucose and other sugars

2. by amino acids and ammonium salts and

3. by phosphate

(Martin, J. F. and A. L Demain: Control of antibiotic biosynthesis, Microbiol., Rev. 44, 230-251 [1980]). In the case of the biosynthesis of streptothricin antibiotic nourseothricin with Spreptomyces noursei IMETJA 3890 b is known that this is adversely affected by amino acids and ammonium salts (Gräfe, U., Steudel, A., Bocker, H. u / id Thrum, H .: Regulatory influence of o-aminobenzoic acid [OABA] on the biosynthesis of nourseothricin in cultures of Streptomyces noursei JA 3890 b.

AIIg. Mikrobiol.20, 185-194 [1980] and literature cited here).

To date, ammonium nitrate has been used as the nitrogen source in the nutrient medium (see Gräfe et al., 1980). In this compound, half of the nitrogen is not present as ammonium ions.

A disadvantage of the technical application is that ammonium nitrate is an explosive and may only be used in conjunction with complex safety measures. Furthermore, so far only at the addition of the relatively costly and polluting inhibitors of respiration and amino acid transport, such as o-aminobenzoic acid (OABA), a maximum of 3000 lb./ml nourseothricin at fermentation end in the culture filtrate of laboratory fermentations can be detected (Gräfe, U., Bocker, H , Reinhardt, G. and Thrum, H .: Regulatory influence on nourseothricin biosynthesis by o-aminobenzoic acid in cultures of Streptomyces noursei JA 3890 b.A., Allig. Mikrobiol., 14, 659-673 [1974]).

It is known that the biosynthesis of a number of secondary metabolites is adversely affected by excess phosphate (Martin, J.F .: Control of antibiotic biosynthesis by phosphate, Adv. Biochem., Eng., 6, 105-7 (1977); Weinberg, D .:

Secondary metabolism: regulation by phosphate and trace elements. Folia Microbiol. 23.496-504 (1978]). Therefore, some technical microbial fermentations for the production of antibiotics are carried out at suboptimal for the growth of the microbial producer phosphate concentrations. In general, complex media components of plant and animal origin, such as starch, soybean meal, corn steep liquor, molasses, meat extract, etc., are used in technical fermentations for the production of secondary metabolites. Depending on their origin and treatment, the total phosphate content and the bioavailability (solubility) of a complex substrate vary.

Different portions of the total phosphate, which is transferred through the complex substrates in the Submersmedium is then present at the start of fermentation as dissolved, immediately available phosphate.

The listed circumstances generally complicate the standardization of the culture media. The bioavailability of the phosphate is of decisive importance for the yield of desired secondary metabolites, because on the one hand a certain amount of phosphate is necessary for the growth of the producing microorganisms and on the other hand too high initial concentrations of soluble phosphate can inhibit the secondary metabolite formation. DD-WP 155239 claims the use of the concentration of phosphate ions contained in the culture medium as a controlled variable for stabilizing a continuous microbial culture. However, the method only relates to the fermentative recovery of biomass, wherein the target value of the phosphate concentration is to be set in the range of 20 to 60 mg / l of phosphorus. The application of this invention to the regulation of biosynthesis of secondary metabolites, especially in microbial batch cultures, is not known.

Regulatory effects of phosphate on nourseothricin biosynthesis are unknown.

Object of the invention

The invention includes the aim of overcoming the disadvantages of previously described Nursütithricin production process and to increase the nourseothricin concentrations in technical fermentations by influencing the fermentation process.

Explanation of the essence of the invention

The invention has for its object to provide a technically favorable process for the industrial production of nourseothricin. Surprisingly, it has been found that in fermentations for the production of nourseothricin after lowering the initial phosphate concentration below a previously found to be optimal value and then adding a corresponding or higher amount of phosphate during cultivation, added after a certain time regime, significantly higher nourseothricin yields can be obtained ,

In addition, it has been found that in a submaxic medium containing CaCO3, upon replacement of the nitrogen source with ammonium nitrate by the sulfate-containing ammonium sulfate in a stoichiometric ratio in terms of nitrogen content, the initial phosphate concentrations are from about 15 mg / l phosphate-P to 1 to 3 mg / l im otherwise unchanged medium is lowered after sterilization with strained water vapor.

In general, the initial concentration of soluble, biologically, immediately available phosphate can be reduced in the sterilized medium by appropriate coordination of sterilization regime and content of sulfate ions and on inorganic salts or metal ions that result in poorly soluble with phosphate or sulfate ion sediments.

As a result of lowering this initial concentration in the presence of a sufficient amount of sulfate ions, a shift in the acidity of the weak acid submerged medium to alkaline pH during sterilization caused by precipitation of CaSO ₄ and the associated removal of physiologically acid sulfate ions from the medium , Accordingly, the initial concentration of soluble or biologically available phosphate is also lowered by the external adjustment of an alkaline pH prior to sterilization. The increase in the pH causes favorable conditions for the precipitation of the phosphate with certain present in the nutrient medium metal ions are given. The precipitation of the phosphate-containing sediments is achieved by the targeted addition of such metal ions, eg. B. of iron (III), aluminum (III), magnesium (II), calcium (II), zinc (II) promoted among others.

Particularly favorable for a phosphate precipitation is the sterilization method used in most industrial fermentations with directly acting tensioned water vapor and the presence of CaCO ₃ , which is used for pH buffering of the fermentations.

The phosphate content of the medium can generally also be lowered by reducing the proportion of phosphate-containing complex substrates. For example, the soybean extraction meal used in nourseothricin fermentation contains about 8 mg of phosphate-phosphorus per gram of meal.

Under the conditions described above, the phosphate initial concentration of dissolved or available phosphate becomes so low that the upper tolerances of the phosphate specific for the nourseothricin-producing microorganisms are in each case undershot. These low phosphate concentrations now offer the possibility of obtaining a phosphate which is largely standardized with respect to the phosphate by means of a defined phosphate feed after sterilization. In addition, the phosphate metabolism of the microorganisms can be controlled via the phosphate addition regime so that a high product yield is obtained. The supply of the phosphate can be carried out in the form of a solution or a solid or a suspension. The phosphate may be present either as free phosphate ions or as chemically or physically bound phosphate. Depending on the feed regime, the supply of phosphate or phosphate-containing substances takes place after sterilization or during the fermentation by a single and / or repeated and / or continuous addition. The continuous addition can be carried out at different rates of addition during the fermentation.

As a boundary condition, it must be ensured that no limitation due to substrates or environmental conditions in connection with the phosphate feed and the resulting growth or increased metabolic activities arise for the product formation. In this way, it is possible to exploit the biological potency of the nourseothricin-forming agent for product formation largely and thus to increase the product yield.

embodiments

At embodiments, the invention will be explained in more detail.

1) Aelyophilic soil-dried spore canning of the NG 13-14 selector of the Nourseothricin-forming strain Streptomyces noursei IMET JA 3890b is spread on a suitable agar medium, for example Emerson agar. After about 7 days of incubation at 30 ⁰ C about 2cm ² pieces of the mycelial lawn are cut out and thus in each case a culture batch of the pre-breeding inoculated. The pre-breeding medium contains per liter of tap water the following starting materials: 40g glucose, 15g deoiled soybean meal, 0.3g KH ₂ PO ₄ , 5g NaCl and 3g CaCO ₃ . The acidity is adjusted to pH 6.5 before sterilization. This medium is filled in 500 ml, sealed with cotton stopper Steil Breast Bottles in quantities of 50 ml. The sterilization is carried out by heating for 35 minutes at 121 ° C in an autoclave. The inoculated Vorzuchtansätze be incubated for 48 hours as shaking cultures at 29 ⁰ C. With 150 ml of the thus obtained Vorzuchten the 2500ml-containing culture mixture of the main culture in the laboratory fermentor is inoculated. For the main culture, a medium is used which contains the following starting materials per liter: 32 g potato starch, 29 g glucose, 11 g de-oiled soybean meal, 11 g (NH ₄ J ₂ SO ₄ , ₂ g MgSO ₄ .7H ₂ 0.1 g NaCl, 6 g CaCO ₃ and 0.5 g ZnSO ₄ .7H ₂ O. 1 ml of Antaphron are added to the silicone-based defoaming agent on 2500 ml of nutrient medium before sterilization, and the pH is 6.0 after sterilization of the fermenter culture vessel filled with 2500 ml at 121 ° C in an autoclave and recooling to 30 ⁰ C, the start-acidity is adjusted by aseptic addition of 10% sterile sulfuric acid of pH 8.4 to pH 6.8. the inoculated fermentation batches 168 are einerTemperaturvon30 hours at ° C and an aeration rate of 2500ml of air per minute with stirring of 800U-min " ^1. From 1.5 hours after inoculation of the main culture, a solution of 4g KH ₂ PO ₄ per liter of distilled water over a period of 5 hours at a rate of 1.8ml / Stun de continuously added by means of a peristaltic pump; This addition then takes place over a period of 21 hours at a metering rate of 3.5 ml / hour. During the fermentation, samples of the culture broth are taken periodically under aseptic conditions and the levels of nourseothricin base determined by the pore diffusion assay using Bacillus subtilis ATCC 6633 as a test, glucose, ammonium nitrogen and solids. The parameters pO ₂ , pH and CO ₂ content in the exhaust air are tracked during the course of the process by recording measurement. The decrease in the acidity of the culture solution below pH 5.8 is prevented by adding sterile 5% sodium hydroxide solution.

When the glucose concentration falls to 5 g / l or the ammonium nitrogen to 0.5 g / l, which is indicated by increasing the measured pH values and the pO ₂ values and lowering the CO ₂ content in the exhaust air, 25 g of glucose or 10 g (NH ₄ ) ₂ SO ₄ as concentrated, sterile solutions added discontinuously.

Further fermentation is carried out without addition of KH ₂ PO ₄ , glucose and (NH ₄ J ₂ SO ₄ under otherwise identical conditions When the fermentation is stopped, the culture solution still has clearly detectable amounts of glucose and ammonium in the two different fermentations Concentrations of nourseothricin base determined in the culture filtrate are given in Table 1. ______________________________________

2) A lyophilically dried on glucose gelatin Myzelkonserve (gross weight of the Konserveninhaltes about 300 mg) of the mentioned in Example 1 Nourseothricin-forming Streptomycetes strain is suspended in 3 ml of a 0.9% sodium chloride solution. From this suspension serve 0.5 ml as inoculum of 400 ml pre breeding of the first stage.

The pre-breeding medium has the same composition and acidity as given in Example 1. This medium is filled into 2 500 ml stoppered bottles closed with cotton plugs in quantities of 400 ml each. Sterilization is carried out by heating for 35 minutes at 121 ^° C. in an autoclave.

The cooled to room temperature and inoculated mixtures are incubated for 48 hours as shaking cultures (180U-min ~ ¹ ) at 29 ⁰ C. With 1200 ml of the culture solution thus obtained of the first Vorzucht a heat sterilized with 150 liters of medium (60 minutes at 121 ⁰ C) of the same composition is filled in the first Vorkulturstufe as the second stage of the Vorzucht, equipped with devices for sterile venting and an agitator stainless steel fermenters (250 Liter gross volume). The cultivation is carried out at 27 ^C C to 29 ⁰ C, a speed of the stirrer of 240 U-min ~ ¹ and aeration rate of 1 liter per liter of culture solution per minute.

Stainless steel fermenters (710 liters gross volume) are used for the main culture and are equipped with devices for sterile aeration and a centrally mounted agitator, each filled with 500 liters of nutrient medium (recipe see example 1). As defoamer 0.5 g / l Antaphron is added. The acidity is adjusted to the pH range of 6.7 to 7.0 before sterilization.

The sterilization of the nutrient medium (without glucose portion) takes place in situ by preheating with jacket steam to about 7O ⁰ C and subsequent heating by direct steam to 121 ⁰ C for 15 minutes. After cooling to about 30 ^° C., the glucose sterilized separately in the form of a 50% strength aqueous solution by heating for 30 minutes at 121 ^° C. is added under aseptic conditions. Prior to inoculation, the starting acidity is reduced from pH 8.6 to between pH 6.9 to 7.1 using sterile 10% sulfuric acid.

The inoculated each with 5% inoculum from the second fermenter Vorzuchtstufe approaches the main culture 120 hours at temperatures between 28 ° C to 30 ⁰ C, an aeration rate of 500 liters of air per minute (0.14 to 0,16MPa pressure) with stirring (320U.min " ¹ ).

One of the two fermentor batches, are from the 2nd to the 32nd hour after the inoculation, an aqueous solution comprising KH contains 87 g / l ₂ PO ₄ metered by means of peristaltic pump at a constant rate so that within 30 hours per liter Culture 40mg of phosphate-P.

During the fermentation, samples of the culture broth are taken periodically under aseptic conditions and in these the concentrations of nourseothricin base are determined by means of the perforated plate diffusion test using Bacillus subtilis ATCC6633 as test germ, on glucose, on ammonium nitrogen and on solids. The parameters pH and CO ₂ content in the exhaust air are tracked during the course of the process by registering measurement.

When the glucose concentration falls below 5 g / l or the ammoniacal nitrogen below 0.5 g / l, recognizable by an increase in the pH values and lowering of the CO ₂ content in the exhaust air, 5000 g of glucose or 1800 g of ammonium sulfate as concentrated, sterile aqueous Solution added discontinuously.

The other fermenter batch receives no additions of phosphate, glucose or ammonium sulfate. A ph regulation does not take place; the continuously measured acidity values range from pH 6.4 to pH 7.2. When the fermentation is stopped, the culture solution still contains amounts above 5 g / l glucose and 0.5 g / l ammonium nitrogen.

The concentrations of nourseothricin base determined in the two different fermentations in the culture filtrate are reproduced in Table 2.

3) A lyophilically dried on glucose / gelatin Myzelkonserve (gross weight of the Konserveninhaltes about 300mg) of a selectant of Nourseothricin-forming strain IMET JA 3890 b is treated with respect to the precultivation as in Example 2).

For the main culture, a laboratory fermentor of 7l gross volume, fitted with sterile aeration devices and a center-mounted agitator, is filled with 4.5 l of nutrient medium.

The cultivation is carried out in a modified medium (Embodiment 1) containing 60 g / L of glucose. As defoamer, 0.5 g of Antaphron NM 40 is added to each culture.

To simulate the technical conditions, the sterilization is carried out in a stainless steel fermentor filled with 2500 l of the nutrient medium without the glucose portion. The acidity is adjusted to pH 7.4 before sterilization. Sterilization is carried out in situ durcl preheating with steam jacket to 75 ° C, followed by heating by means of direct steam at 120 ⁰ C for 15 minutes. After cooling to 29 ° C, the strength in the form of a 50% solution by heating to 120 ⁰ C 30rrinütiges separately sterilized glucose is added under aseptic conditions. Before inoculation, the starting acidity is lowered with sulfuric acid of pH 8.1 so that after inoculation the initial value is at pH 7.4. The fermentation batch is inoculated with 4% inoculum from the second pre-breeding stage. To carry out the experiment, 4.51 are transferred from the fermenter to the laboratory fermenter, which is autoclaved separately in the unfilled state.

The main culture lasts for 146 hours at a temperature of 29 ° C and a ventilation rate of 4.51 air per minute. _The: start speed of the stirrer of 800U · min ^"1 is increased to achieve a better oxygenation of the culture, from the 28th hour the stirrer speed 1000 r · min ^'1. Between the 2nd-40th One hour after inoculation, 784 mg of KH ₂ PO ₄ are metered in as aqueous solution by means of a peristaltic pump with time-variant rates of between 1.25 and 1.75 mg of phosphate-P per hour per liter of culture. The automatic addition of 25% aqueous ammonia solution ensures that the control threshold, pH 6.2, is not fallen short of, and thus also an adequate supply of ammonium nitrogen to the culture. Glucose is discontinuously added as a concentrated aqueous solution whenever the process-accompanying analysis indicates the drop in the glucose concentration below 10 g / l. During the fermentation, samples of the culture solution are taken periodically under aseptic conditions and the concentration of the nourseothricin base in the culture filtrates is determined by means of a perforated plate diffusion test. (Table 3).

-4- * »/ o / υ

4) In a fermentation carried out analogously to Example 3), the zinc sulfate fraction of the main culture medium is replaced by 0.2 g / l of Fe ₂ (SO ₄ I ₃ .9H ₂ O. The nourseothricin concentration in the culture filtrate is 8200 μg / ml after 120 hours of fermentation (Tab. 4).

5) In a fermentation carried out analogously to working example 3), the proportion of zinc sulphate is replaced by 0.13 g / l Al ₂ (SO 4 .3H ₂ O. The nourseothricin concentration in the culture filtrate is 8400 / xg / ml after 120 hours of fermentation (Table 4) ).

6) In a further fermentation carried out as far as Example 5), the content of the submerged medium is reduced from 11 g / l de-oiled soybean to 4 g / l. Furthermore, the pH of the medium is adjusted to pH 8.1 with dilute sodium hydroxide solution before sterilization. After sterilization with strained steam, the pH is adjusted with concentrated sulfuric acid so that it is at pH 7.2 after inoculation. The phosphate metering is carried out between the 2nd to the 72nd hour by an aqueous KH ₂ PO ₄ solution which is added at a constant rate in such a way that a phosphate supply of 1.4 mg of phosphate-P per hour and liter of culture is achieved. During the fermentation, glucose is added discontinuously so that the glucose concentration does not fall below 1 g / l. The pH is maintained at pH 6.0 by titration with 25% ammonium hydroxide solution.

The achieved nourseothricin concentration are listed in Table 5.

Table 1: Nourseothricin contents in KH ₂ PO ₄ , glucose and (NH ₄ J ₂ SO ₄ dosed and in non-dosed laboratory fermenter cultures of the strain Streptomyces noursei IMETJA3890b - // NG 1-3-14 as a function of the fermentation time

Fermentation time with dosage without dosage (control)

h / ig / ml% / ig / ml%

Table 2: Nourseothricin contents in 5001 fermenter cultures of the strain Streptomyces noursei dosed with HK ₂ PO ₄ , glucose and (NH ₄ J ₂ SO ₄ and not dosed IMET YES 3890b - // NG 13-14 as a function of the fermentation time

Fermentation time with dosage without dosage (control)

hjag / ml% / lig / ml%

Table 3: Nourseothricin content in a 4.51 fermentor culture of a strain of Streptomyces noursei strain IMET JA 3890b, dosed with KH ₂ PO ₄ , glucose and (NH ₄ I ₂ SO _4, depending on the fermentation time

1035 249 415 100 2980 165 1805 100 5412 294 1840 100 8311 213 3905 100 8800 195 4520 100 9230 213 4005 100

225 216 104 100 1600 228 702 100 4858 299 1627 100 4903 208 2362 100 6862 191 3585 100 7781 200 3885 100

fermentation time 20 Nourseothricinkonzentration H 44 Mg / ml 68 320 92 2330 116 6240 146 8010 11040 14070

Table 4: Nourseothricin contents of a corresponding Ausεführungsbeispiel 4) with Fe (III) ions and according to embodiment 5) with Al (III) ions carried out fermentation in dependence on the Fe. wüsntationszeit

Fermentation time Nourseothricin concentration μg / ml in the embodiment

h 4) 5)

530 800 2500 3200 5400 6300 7250 7400 8200 8400

- ο - ** / »» ι \ ι

Table 5: Nourseothricin contents of a laboratory fermenter culture dosed with KH ₂ PO ₄ , glucose and ammonium hydroxide and containing Al (III) ions as a function of the fermentation time

fermentation time 24 Nourseothricmkonzentration H 48 // g / ml 72 800 96 2000 120 5660 8900 11000 *

Claims (1)

- I - t / O IV Invention claim: Process for the fermentative production of nourseothricin by adjusting the concentration of biologically available phosphate, characterized in that measures applied individually or in combination are applied by: The sterilization of the nutrient media with strained steam in the presence of metal ions which form sediments which are sparingly soluble with phosphate, after the acidity of the nutrient media has been adjusted to values between pH 7 to pH 9 and / or simultaneous presence of sulfate ions and calcium carbonate, The content of the nutrient media on phosphate-containing complex substrates is lowered before sterilization, - Phosphate is fed to the beginning of fermentation and / or during the further course of the fermentation while avoiding further limitations.