GB2075056A

GB2075056A - L-proline-producing Microorganisms

Info

Publication number: GB2075056A
Application number: GB8111730A
Authority: GB
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 1980-04-14
Filing date: 1981-04-13
Publication date: 1981-11-11
Also published as: FR2480307A1; FR2480307B1; JPS56144093A; GB2075056B

Abstract

L-proline-producing microorganisms are constructed by introducing, into a recipient strain of the genus Escherichia, a hybrid plasmid having had inserted therein a DNA fragment possessing genetic information relating to L-proline production and derived from a donor strain of the genus Escherichia, which donor strain is resistant to a proline- analogue.

Description

SPECIFICATION L-proline-producing Microorganisms This invention relates to L-proline-producing microorganisms constructed by a gene recombination technique.

Most wild microorganism strains do not produce L-proline. In order to render a wild strain capable of producing L-proline from carbohydrates, it is necessary to induce artificial mutants from the wild strain. There are many known proline-producing artificial mutants. Typical known prolineproducing mutants are isoleucine and arginine-requiring mutants of Brevibactrerium flavum (U.S.

Patent No. 3,329,577), isoleucine-requiring mutants of Micrococcus glutamicus (British Patent No.

1,172,903), mutants of Kurthia catenaforma (British Patent No. 1,186,270), mutants of Microbacterium ammoniaphilum (Publication of Japanese Examined Patent Application No.

38876/1 973), mutants resistant to sulfa-drugs, of the genus Brevibacterium and Corynebacterium (U.S. Patent No. 3,81 9,483), tyrosine and phenylalanine-requiring mutants of Corynebacterium melassecola (Publication of Japanese Examined Patent Application No.33190/1976), homoserinerequiring mutants of the genus Corynebacterium (Publication of Japanese Unexamined Patent Application No. 41386/1979), and mutants resistant to 3,4-dehydroproline, of the genus Brevibacterium and Corynebacterium (Publication of Japanese Unexamined Patent Application No.

105293/1979).

It is further known that a particular mutant of Escherichia coli excretes L-proline [Biochem, Biophys. Acta, 104,395 (1965)]. The most efficient proline-producer, as far as we know, is Brevibacterium flavum FERM-P 4371 which produces 3.6 g/dl of L-proline from 10 g/dl of glucose. It has, however, become difficult to increase the yields of L-proline using artificial mutation techniques.

According to the present invention, there is provided an L-proline-producing microorganism obtained by incorporating, into a recipient strain of the genus Escherichia, a hybrid of plasmid having had inserted therein a DNA fragment possessing genetic information relating to L-proline production, which fragment is derived from a donor strain of the genus Escherichia resistant to a proline-analogue.

The present invention also provides a process for producing L-proline, which comprises culturing an L-proline-producing microorganism of the invention.

The DNA-donor strain used to construct the L-proline producer of this invention is a mutant of the genus Escherichia resistant to a proline-analogue and capable of producing L-proline. Strains having a higher productivity of L-proline are preferably used as the DNA-donor. The L-proline-analogue used in this invention inhibits the growth of Escherichia strains, which inhibition is, however, suppressed when L-proline coexists in the medium. Examples of the proline-analogue are 3,4-dehydroproline and azetidine-2-carboxylic acid.

In order to increase the L-proline-productivity of the mutant, it is effective to make the mutant require for growth an amino acid such as isoleucine, histidine, ornitine, arginine, methionine and leucine. The L-proline productivity is also increased by giving the mutant a resistance to sulfa-drugs such as sulfa-guanidine.

Chromosomal DNA is extracted from the DNA donor in a known manner and treated with a restriction endonuclease by a known method [see, for example, Biochem. Biophys. Acta, 383, 457 (1975)].

The plasmid or phage DNA used as the vector in the synthesis procedure is also treated with a restriction endonuclease in an analogous manner. Various restriction endonucleases can be used, if the digestion of the chromosomal DNA is effected partially. Thereafter, the digested chromosomal DNA and vector DNA are subjected to a ligation reaction. Recombination of the DNA to prepare the recombinant plasmid can be carried out by introducing, by the use of terminal transferase, deoxyadenylic acid and deoxythymidylic acid, or deoxygualylic acid and deoxycytidylic acid, into the chromosomal DNA fragment and cleaved vector DNA, and by subjecting the modified chromosomal DNA fragment and the cleaved vector DNA to an annealing reaction.

As a suitable vector DNA, a conventional vector can be employed, such as Col El, pSC 101, pBR 322, pACYC 177, pCR 1, R6K or A-phage, or their derivatives.

The hybrid DNA thus obtained can be incorporated into a microorganism of the genus Escherichia by conventional transformation techniques [see, for example, J. Bacteriol., 119, 1072 (1974)]. The desired transformant is screened by using a medium on which only a clone having one or both of the characteristics of L-proline productivity originating from the chromosomal DNA fragment and from the vector DNA, can grow.

As the recipient microorganism for the hybrid DNA, an L-proline-auxotroph is usually used, since it is conventional to distinguish the proline-producing transformant from the recipient. Desirably, a mutant already having a higher productivity of L-proline is used as the recipient, to obtain better results.

The methods of culturing the L-proline-producing strains thus obtained are conventional, and are similar to the methods for cultivation of known L-proline-producing microorganisms. Thus, the culture medium employed may be a conventional one containing carbon sources, nitrogen sources, inorganic ions and, when required, minor organic nutrients such as vitamins or amino acids. Examples of suitable carbon sources include glucose, sucrose, lactose, starch hydrolysate and molasses. Gaseous ammonia, aqueous ammonia, ammonium salts and other nitrogen containing materials can be used as the nitrogen source.

The cultivation of the recombinant microorganisms is usually conducted under aerobic conditions, the pH and the temperature of the medium being adjusted to a suitable level, and may be continued until the formation of L-proline ceases. The L-proline in the culture medium can be recovered by conventional procedures.

By the method of the present invention, L-proline can be produced in higher yields than has been achieved in previously known methods in which artificial mutants of Escherichia are used.

The invention will now be illustrated by the following Example.

Example (1) Preparation of Chromosomal DNA Possessing Genetic Information Relating to L-proline Production Escherichia coli EG-19 (NRRL B-i 2391), a mutant resistant to S-(2-aminoethyl)-cysteine and 3,4-dehydroproline, and induced from K-12 (ATCC 10798), was cultured at 370C for 3 hours with shaking in 1 litre of L-medium containing 1 g/dl of peptone, 0.5 g/dl of yeast extract, 0.1 g/dl of glucose and 0.5 g/dl of NaCI (pH adjusted to 7.2), and bacterial cells in the exponential growth phase were harvested. Chromosomal DNA was extracted by a conventional phenol-method, whereby 3.4 mg of purified DNA were obtained.

(2) Preparation of Vector DNA As the vector, the DNA of plasmid pBR 322, which contains both ampicillin and tetracycline resistant genes as makers, was prepared as follows. A strain of Escherichia coli K-12 harbouring the plasmid pBR 322 was incubated at 370C in 1 litre of a glucose-"casamino acid"-inorganic salts medium containing 2 g of glucose, 1 g of NH4CI, 6 g of Na2HP04, 3 g of KH2P04, 5 g of NaCI, 0.1 g of MgS04, 7 H20, 0.01 5 g of CaCI2 2 2 H20, 2 g of "casamino acid", and 100 ,ug of thiamine HCI, each per litre (pH adjusted to 7.2). After the strain had been incubated until the late 10 g or log phase, 170 ,ug/ml of chloramphenicol were added to the culture medium. By this process, the plasmid DNA was amplified and accumulated abundantly in the bacterial cells.

After 16 hours of incubation, the cells were harvested and then lysed by treatment with lysozyme and sodium docecylsulphate (SDS). The lysate was centifuged at 30,000 xg for 1 hour to obtain a supernatant. After concentrating the supernatant, 480 ,ug of the plasm it DNA was obtained by fractionation using cesium chloride-ethidium bromide equilibrium density gradient centrifugation.

(3) insertion of the Chromosomal DNA Fragment into the Vector Portions, each of 10 yg, of the chromosomal DNA were treated with the restriction endonuclease Hind Ill at 370C for 5, 10, 20, 30 and 60 minutes, respectively, to cleave DNA chains, and each portion was then heated at 650C for 5 minutes. Portions, each of 10 jug, of the vector DNA were also treated with the restriction endonuclease Hind Ill at 370C for 1 hour to cleave the DNA completely, and then were heated at 650C for 5 minutes.

The digested chromosomal DNA solution and cleaved vector DNA solution was mixed and subjected to a ligation reaction for DNA fragments by the use ofT4 phage DNA-ligase in the presence of ATP and dithiothreitol at 100C for 24 hours. The reaction mixture was then heated at 650C for 5 minutes, and a two-fold volume of ethanol was added to it. The recombinant DNA which precipitated was recovered.

(4) Genetic Transformation With the Hybrid Plasm it Harbouring Genetic Information Relating to Proline Production Mutant strains No. 19 (NRRL B-12394) and No. 30 (SG) (NRRL By12396), which are resistant to S-(2-aminoethyl)cysteine and sulfaguanidine, respectively, and which were derived from Escherichia coli K-i 2 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, were cultured in 10 ml of L-medium at 370C with shaking. Cells in the exponential growth phase were harvested, and suspended in a 0.1 M MgCI2 solution and then in a 0.1 M CaCI2 solution in an ice-bath, whereby "competent" cells having the ability of DNA uptake were prepared.

Into the competent cell suspension, the DNA obtained in step (3), which contains the hybrid plasmid DNA, was added. The suspension was kept in an ice-bath for 30 minutes, then heated at 420C for 2 minutes, and again allowed to stand in an ice-bath for 30 minutes. The cells, thus containing the hybrid plasmid DNA, were inoculated into an L-medium and the medium was shaken at 37"C for 3 hours whereby the transformation reaction was complete. The cells were harvested, washed, and resuspended. A small portion of the cell suspension was spread on an agar plate containing 2 g of glucose, 1 g of (NH4)2SO4,7 g of K2HPO,2 g of KH2PO4, 0.1 g of MgSO4 .7 7 H2O, 0.5 g of sodium citrate .2 2 H20, 0.5 g of S-(2-aminoethyl)-L-cysteine . HCI and 2 g of agar, each per litre, and further containing 20 yg/ml of ampicillin (pH adjusted to 7.1). The plate was incubated at 37 OC. After 3 days incubation, all of the colonies which appeared were picked up, purified and isolated.

Colonies which were resistant to both ampicillin and S-(2-aminoethyl)-L-cysteine and which had the ability of producing L-proline were obtained as transformants. Resistance to ampicillin (50 4g/ml) and to S-(2-aminoethyl)-L-cysteine (500 Mg/ml) were tested using an agar-L-medium, and L-proline productivity was examined by cultivation at 31 0C for 72 hours. Transformants AJ 1 1543 (FERM-P 5483, NRRL B-12403) and AJ 11 544 (FERM-P 5484, NRRL B-12404) were obtained from recipient strains No. 1 9 and No. 30 (SG) respectively.

(5) Production of L-proline by the Proline-producing Strains The table below shows the experimental results of the fermentative production of L-proline using the L-proline-producing strains AJ 1 1543 and AJ 11544, and the strains EG-19, No. 19 and No. 30.

The fermentation medium contained 5 g/dl of glucose, 2.5 g/dl of ammonium sulphate, 0.2 g of KH2PO4,0.1 g/dl of MgS04. 7 H20, 0.05 g/dl of yeast extract, 1 mg/l of thiamine. HCI, 1 mg/dl of FeSO4. 7 H20, 1 mg/dl of Mn SO4. 4 H20 and 2.5 g/dl of CaCO3 (separately sterilized), and its pH was adjusted to 7.0. Batches, each of 20 ml, of the fermentation medium were placed in 500 ml flasks and inoculated with one loopful inoculum of the test microorganisms, and cultivation was performed at 31 OC for 72 hours.The amount of L-proline in the supernatant of the fermentation broth was determined by microbiological assay.

Table

Amount of L-proline Microrganism tested produced {mg/dl) EG-19 0.8 No. 19 0 No. 30 0 At 11543 15.0 AJ 11544 25.0 The above-mentioned strains having NRRL-numbers have the same taxonomic characteristics as strain K-12, which in turn has the taxonomic characteristics given in "Bergey's Manual of Determinative Bacteriology" 8th Edition, and they were deposited at the Agricultural Research Culture Collection (NRRL) (an International Depositary Authority under the Budapest Treaty) on the 1 itch March 1981.

Claims

1. An L-proline-producing microorganism obtained by incorporating, into a recipient strain of the genus Escherichia, a hybrid plasmid having had inserted therein a DNA fragment possessing genetic information relating to L-proline production, which fragment is derived from a donor strain of the genus Escherichia resistant to a proline-analogue.

2. A microorganism as claimed in claim 1, wherein said recipient strain is of Escherichia coli.

3. A microorganism as claimed in claim 2, wherein said recipient strain is Escherichia coli K-12 or a mutant thereof.

4. A microorganism as claimed in any of claims 1 to 3, wherein said donor strain is of Escherichia coli.

5. A microorganism as claimed in claim 4, wherein said donor strain is Escherichia coli K-i 2 or a mutant thereof.

6. A microorganism as claimed in any of claims 1 to 5, wherein said hybrid plasmid is derived from pBR 322.

7. Escherichia coliAJ 11543 (NRRL B-12403).

8. Escherichia coli AJ 11544 (NRRL B-12404).

9. A process for producing L-proline, which comprises culturing an L-proline-producing microorganism as claimed in any of claims 1 to 8.

10. A process according to claim 9, substantially as described herein.

11. L-proline produced by a process according to claim 9 or 10.