FR2556009A1 - STRAIN OF AN L-PROLINE PRODUCING MICROORGANISM, PROCESS FOR OBTAINING L-PROLINE PRODUCING MICROORGANISMS AND PROCESS FOR PRODUCING L-PROLINE - Google Patents

Abstract

THE INVENTION IS RELATED TO THE PRODUCTION OF L-PROLINE. ACCORDING TO THE INVENTION, THE PRODUCTION OF L-PROLINE IS IMPROVED WHEN A MULTIPLE ANALOGUE RESISTANT MICROORGANISM STRAIN OF PROLINE IS OBTAINED; AN IMPROVED BREVIBACTERIUM AMMONIAGENES STRAIN, DESIGNATED BY ATCC N39101 HAS BEEN ISOLATED; THIS STRAIN IS RESISTANT TO N-AZETIDINE-2-CARBOXYLIC ACID AND 3,4-DEHYDRO-DL-PROLINE, HAS A MODIFIED GENETIC STRUCTURE WITH RESPECT TO THE L-PROLINE REACTION MECHANISM AND HAS BEEN SHOWN TO PRODUCE L-PROLINE IN COMMERCIAL QUANTITIES. THE INVENTION APPLIES IN PARTICULAR TO THE PRODUCTION OF AMINOACIDS.

Description

The production of L-proline (here called "proline") and other amino acids

  fermentation has been the subject of considerable research. Microorganisms belonging to the genera Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium and others, require a certain amount of proline in order to survive and will produce their own proline. Such proline production does not necessarily depend on a lack of proline in the medium, indeed some microorganisms can still produce certain amounts of proline. Nevertheless, it is believed that microbial production of proline is controlled, at least partially, by certain reaction mechanisms such that the microorganisms usually cease or reduce the production of proline when they dispose of it.

in sufficient quantities.

  When an analogue of proline is present, the

  microorganisms generally recognize it and use it

  as proline, thereby triggering the reaction mechanism to stop or slow down the production of

  proline. In the absence of proline produced in the environ-

  internally, microorganisms die.

  Some mutants are insensitive or resistant to proline and proline analogues. The reaction mechanism of these mutants is not triggered by either proline or proline analogues. As a result, these mutants will continue to produce more proline than necessary. The patent and technical literature contain numerous references describing resistance to proline analogs as a means for selecting overproducing proline microorganisms. For example, Japanese Patent Publication No. 55-148096 discloses a method of producing proline by culturing microorganisms belonging to the genus Corynebacterium, Arthrobacter, Brevibacterium or Microbacterium, which are resistant to proline structural analogues such as , 4-deh-ydroproline, hydroxyproline, azitidine-2-carboxylic acid, proline hydroxamate and D-proline. Similarly, U.S. Patent No. 4,244,409 to Nakamori et al., Discloses a method of producing proline, using mutants belonging to the genus Brevibacterium, Corynebacterium or Microbacterium,

  and which are resistant to DL-3,4-dehydroproline. The micro-

  proline analog-resistant organisms are typically obtained by exposing known proline producers or known producers of glutamic acid to a-mutagen and then sorting mutants that can produce

  proline in the presence of the proline analogue.

  It has been found that unique microorganisms characterized by double resistance to proline analogues are useful for the production of proline in commercial quantities. More particularly, a strain of Brevibacterium ammoniagenes has been identified which can produce considerably more proline than either the non-resistant parent strain or a single analog-resistant intermediate strain. The new strain is resistant to both L-azetidine-2-carboxylic acid ("ACA") and 3,4-dehydro-DL-proline ("DHPI"). The proline produced by culturing microorganisms of this new strain can be easily separated from the

  fermentation broth by standard methods.

  The present invention relates to the production of a new microorganism capable of better

  production of proline in commercial quantities.

  Another object of the present invention is a

  organism where the genetic structure has been modified to make the microorganism resistant to multiple

proline analogues.

  In addition, it is desirable that the new strain be useful in commercial processes.

  fermentation standards or their improvements.

  The present invention is directed to novel strains of microorganisms whose modified genetic structure renders the microorganisms resistant to two proline structure analogs, ACA and DHP, or

  allows them to resist. As a result, micro-

  organisms will produce excessive amounts of proline when cultured under aerobic conditions. According to one embodiment, a new strain has been derived from a culture of Brevibacterium ammoniagenes obtained at

  the American Type Culture Collection under the N ATCC 13746.

  Brevibacterium ammoniagenes ATCC 13746 is a known producer of glutamic acid which has been found in preliminary screening to produce small amounts of proline (less than one milligram per liter). It was

  desirable to obtain, by resistance to multiples -

  structural analogues of proline, a mutant strain that can produce significantly elevated levels of proline. The method of selection was to expose ATCC 13746 strain to ACA and to look for a mutant

  spontaneous with resistance to ACA. This intermediate mutant

  ACA-resistant diary was exposed to DHP and a mutant was found with the desired characteristic, resistance

multiple analog alux.

  Nutrient Slant Agar (marketed by Difco) of ATCC 13746 was washed with sterile water

  deionized. A 0.2 ml portion of the resulting suspension

  The cells were plated on a plate of agar or nutrient agar (Difco) containing 10 mg / ml of the proline analog, ACA. This plate was incubated at 30 ° C. for 3 days. Due to the presence of ACA in the culture medium, the prototrophic growth of ATCC 13746 was suppressed and only ACA-resistant microorganisms were able to grow. The largest single resistant colonies growing on the ACA plate were transferred to a fresh nutrient agar plate (Difco) without ACA. This plate was incubated until the ACA resistant colonies

have grown up, about 3 days.

  One of these ACA-resistant clones was found to produce high levels of proline during growth

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  in Proline Medium C, contents of which are as follows: Ingredient Amount (per liter deionized water) Glucose 50, Q g NH4Cl 5.0 g Urea 5.0 g KH2PO4 0.5 g K2HPO4.5 g MgSO4 7 H 2 O 0.5 g FeSO 4 7 H 2 O 0.02 g MnSO 4 4 H 2 O 0.02 g ZnSO 4 - 7 H 2 O 0.01 g Biotin 100.0 g Thiamine-HCl 1.0 mg The pH of the medium was adjusted to neutrality , about 6.8, with soda and the middle was passed to

  autoclave for 12 minutes at 112 C.

  The ACA-resistant clone in ml of Proline Medium C was grown in a serrated Erlenmeyer flask for 3 days at 30 ° C and 300 rpm. The broth was collected, centrifuged at 4000 rpm and

  filtered through a 0.22 micron millipore filter.

  The proline titer was determined by the Technicon II self-analyzer method using isatin dye. The process followed was to heat 1%

  Isatin dye in isopropanol with the sample

  lon for 1 minute at 80 C then to determine the absorption at 590 nm. ACA-resistant clone was found

produced 8.5 mg / ml of proline.

  This ACA-resistant intermediate mutant was inoculated on an agar slant of nutrient agar (Difco) and incubated for 4 days at 30 ° C. The slant agar was washed with 5.0 ml of sterile deionized water. A 0.2 ml portion of the resulting cell suspension was plated on a nutrient agar plate (Difco) containing 2 mg / ml DBH. This plate was incubated at

 C for 3 days.

  One of the colonies growing on this plate, resistant to both ACA and DHP, was transferred to a fresh plate of nutrient agar (Difco) containing neither ACA nor DHP. This strain was then grown in a Proline C10S medium, the contents of which are as follows: Ingredient Amount (per liter of deionized water) Glucose 100.0 g NH4Cl 5.0 g Urea 5.0 g KH2PO4 0.5 g K2HPO4 0.5 g MgSO4-7 H20 0.5 g FeSO4.7 H20 0.02 g MnSO4 · 4 H20 0.02 g ZnSO4.7 H20 0.01 g Bacto-Soytone (Difco) 0.3 g Biotin 100 , 0. Lg Thiamine-HCl 1.0 mg The pH of the medium was adjusted to neutrality, about 6.8, with sodium hydroxide, and the medium was stirred.

  autoclaved for 12 minutes at 112 C.

  One of the ACA and DHP-resistant clones was grown for 3 days in 50 ml of a Proline C10S medium in a serrated Erlenmeyer flask. The proline titer was determined by isatin dye as described above. The clone was found to produce proline at levels of 19.6 mg / ml. A freeze-dried culture of this newly developed resistant strain of ACA and DLP of Brevibacterium afmmioniagenes was deposited at the American Type Culture Collection, 12301 Parklawn. Drive, Rockville, Maryland, 30852, United States of America, without restrictions

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  the availability to the public at the grant of this patent.

  The culture was designated ATCC N 39101.

  Another fermentation using ATCC 39101 for proline production was made using the following media: Seed medium Ingredient Amount Glucose 5.0 g NaCl 5.0 g Yeast extract 10.0 g Peptone 10.0 g Ingredients were mixed in deionized water to 1 liter, the pH was adjusted to 7.0 with sodium hydroxide and the medium was autoclaved for

minutes at 112 C.

  - Production medium Ingredient Quantity 1 Urea 5.0 g NI4Cl 5.0 g K2-24 - 1.0 g MgSO4 7H20 0.5 g 4O 2 Saca isolate (Sheffield Co.) 1.0 g FeSO4 7H20 1.0 ml MnSO4 H20 2.0 ml 3 ZnSO4 7H20 1.0 ml Biotin 1.0 ml Thiamine-HCl 1.0 ml6 CaCO3.50.0 g Glucose 7 1. The addition of glucose should be taken into consideration when

  the total volume of deionized water is determined.

  2. Supply solution: 20.0 g / l FeS04'7H20 3. Supply solution: 6.1 g / l MnSO4.H20 4. Supply solution: 10.0 g / l ZnSO4.7H20

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  5. Supply solution: 0.1 g / l of biotin 6. Supply solution: 1.0 g / 1 of thiamine-HCl

  7. Supply solution: 70% glucose.

  Autoclave at 110 C for 15 minutes. Add 7.0 ml / flask before inoculation. The first ten ingredients were mixed with deionized water to form 1 liter, the pH was adjusted

  at 6.8 with sulfuric acid and CaCO3 was added.

  The medium was autoclaved at 121 ° C. for

15 minutes.

  A 250 ml baffled flask containing 50 ml of the seed medium was inoculated with Brevibacterium ammoniagenes ATCC 39101 and incubated at 31 C, 300 rpm for 24 hours. About 5.0 ml of the log phase culture (0.D.640 = 3.0) was sonicated to disrupt the cell clusters. The ultrasonic cell suspension was diluted with sterile 0.9% NaCl solution and plated on nutrient agar (Difco) to obtain single colonies. The plaques

  were incubated at 30 C for 72 hours.

  A single colony of the plates was inoculated into a 250 ml baffled flask containing 50 ml of the seed medium and incubated at 31 ° C. at 300 rpm for 16 hours. At this time, the seed culture was in the early growth stage (0 D640-1.7). The seed culture was used to inoculate 250 ml baffled flasks containing

  millet production medium to an initial OoD.640 of 0.05.

  O. each balloon: 50 # cl of 25.0% of Pluronic 61 HQ 'VB, Wvyandotte) was added as anti-foam. The productiorh flasks were incubated at 310C, 300 rpm. A high precision liquid chromatographic analysis of a sample of oil collected after 48 hours.

  has shown proline levels e 15.8 g / l.

  The microorganisms of this strain can be used to produce proline by any arobie method: nvenetial onalture or fermentation.

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  The fermentation is typically carried out at 20-45 ° C, preferably at 2835 ° C and at pH 5 to 9. Calcium carbonate and ammonia may be used to

adjusting the pH of the medium.

  The culture medium may be any fermentation medium containing a carbon source, a nitrogen source, inorganic salts and, if desired, other minor organic foods. As a carbon source, fermentable sugars, protein hydrolysates and proteins can be used. As a nitrogen source, urea, ammonium salts of organic acids (such as ammonium acetate or ammonium oxalate) and ammonium salts of inorganic acids (such as ammonium, ammonium nitrate or ammonium chloride) may be satisfactory. The amounts of carbon and nitrogen sources in the

  medium are comprised between 0.001 and 20 w / v%.

  Inorganic elements (such as potassium phosphate or magnesium sulfate) and / or vitamins (such as

  thiamine) can also be added.

  Biotin, a vitamin B complex, is a nutrient necessary for culturing Brevibacterium ammoniagenes ATCC 39101. The concentration of biotin in the culture medium may range from about 50 to about 500 g / l. Concentrations

  below this range tend to force this micro-

  organism towards the production of glutamic acid, which is an undesired contaminant when proline is

  and the amino acid that one seeks to produce.

  The addition of other organic nutrients

  in the culture medium may be desired. The microorganism according to the invention is considered to be a bradytrophe, i.e. a slowly growing microorganism, which will develop more probably when grown with complex nutrients than on a minimum medium. For example, organic nutrients (such as

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  soaking of mals, peptones, hydrolysates of

  protein or yeast extracts), inor-

  ganes (such as potassium phosphate or magnesium sulphate) and / or vitamins (such as thiamine) can be added. The preferred culture medium for microorganisms of this type must contain organic nutrients. One of the preferred organic nutrients for fermentation at the flask is BactoSoytone (Difco), an enzyme hydrolyzate of soy flour, at a concentration of about 0.1 to about 0.5, preferably about 0.3 gram per liter of culture medium. Alternatively, yeast extracts, quench liquors or peptones from other sources may be used. In addition, the

  Vitamin Thiamine is a favorite nutrient.

  The fermentation is accomplished in about 16 to 176 hours, typically 72 hours at the flask, and during this time the proline accumulates in the fermentation broth. The cells and other solid components of the culture can be removed from the broth by conventional processes such as

filtration or centrifugation.

  Known processes can be used for the recovery and / or purification of proline from the filtrate or supernatant solution. For example, the filtered fermentation broth can be processed using an ion exchange resin. Proline can

  be crystallized from the resulting solution.

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Claims (11)

R E V E N D I C A T I 0 N S   1. A strain of an L-proline producing microorganism, characterized by resistance to analogs   multiples of proline and by the ability to   production of L-proline. 2. A strain according to claim 1, characterized in that the microorganism is resistant to two or more proline analogues selected from the group consisting of 3,4-dehydro-DL-proline, azetidine-2-acid. carboxylic acid, proline hydroxamate and D-proline.   3. A strain according to claim 2, characterized in that the microorganism is a strain of   - Brevibacterium ammoniagenes and is resistant to 3,4-dehydro-   DL-proline and L-azetidine-2-carboxylic acid.   4. A strain according to claim 3, characterized in that the microorganism is Brevibacterium ammoniagenes ATCC 39101.   The strain of claim 1, further characterized in that a nutrient medium comprising carbon and nitrogen sources in amounts of from about 0.001 to about 20.0 weight is used. volume%.   6. A strain according to claim 5, characterized in that the nutrient medium comprises biotin in an amount of about 50 to about 500 micrograms per liter.   7. A strain according to claim 5, characterized in that the nutrient medium comprises at least one complex nutrient selected from the group consisting of peptones, protein hydrolysates, extracts   yeast and soaking liquors of mals.   8. A process for obtaining microorganisms allowing improved production of L-proline, characterized in that it consists in: (a) exposing L-proline-producing microorganisms to a first proline analogue, (b) growing first proline resistant colonies which survive the exposure of step (a); (c) exposing said first proline analog resistant colonies to a second proline analog; , and (d) grow the first and second proline-resistant colonies that survive the exposure of step (c).   9. A process according to claim 8, characterized in that the first proline analogue and the second proline analog are selected from the group   consisting of 3,4-dehydro-DL-proline, azetidine   2-carboxylic acid, proline hydroxamate and D-proline, and that the first proline analogue and the second proline analog are not the same analog of proline.   10. A process for the production of L-proline, characterized in that it consists in: (a) choosing a strain of L-proline-producing microorganisms which is resistant to multiple proline analogues, (b) culturing or in fermentation said strain in culture medium comprising a carbon source and a nitrogen source, and   (c) recovering L-proline from the culture medium.   Process according to claim 10, characterized in that the strain of the oriol-producing microorganisms is a strain of Brevibacteri anmmoniagenes. two different analogues of proline   u. is in the group consisting of 3,4-dehydroDL;   King L-aztcidine-2-carboxylic acid, hydroxamate proline and D-proline.   12. A process according to claim 10, characterized in that the culture medium comprises at least one complex nutrient selected from the group consisting of peptones, protein hydrolysates,   yeast extracts and soaking liquors of mals.   13. A process according to claim 11, characterized in that the strain of L-proline producing microorganisms is Brevibacterium ammoniagenes ATCC 39101.