DE2839052A1 - MICROBIOLOGICAL PROCEDURE - Google Patents

Description

The invention relates to a microbiological process, in particular to the production of single-cell protein (SCP) from methanol by cultivating genetically modified bacteria, to such bacteria and to a process for their production.
20th

In the production of protein by the microbiological assimilation of methanol and ammonia, two mechanisms for ammonia assimilation have been identified, which are given below:
25th

(a) direct (GDH)

HO ₂ C- (CH ₂ J ₂ -CO-CO ₂ H + NH ₃ + NAD (P) H Cfr -ketoglutarate

- HO ₂ C- (CH ₂ ) ₂ -CH-CO ₂ H + NAD (P) 30

NH ₂ glutamate

This reaction is catalyzed by the enzyme glutamate dehydrogenase (GDH) and consumes 1 molecule of NAD (P) H, which is 3 molecules ATP is equivalent to glutamate per molecule of the product.

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Deutsche Bank (Munich) Account 51/61070 Dresdner Bank (Munich) Account 3939 844 Postscheck (Munich) Account 670-43-804

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(b) two levels (GS-GUS)

HO ₂ C- (CH ₂ ) ₂ -CH-CO ₂ H + NH ₃ + ATP ^ H ₂ NCO- (CH ₂ ) ₂ -CH CO ₂ H + ADP + Pi

NH ₂ NH ₂

'Glutamate Glutamine

This reaction is triggered by the enzyme glutamine synthetase (GS) catalyzed.

HO ₂ C (CHj) ₂ -CO-CO ₂ H + H ₂ -NCO (CH ₂ ) ₂ -CH-CO ₂ H + NAD (P) H

NH ₂ ir ** 2 HO ₂ C (CHj) ₂ -CH-CO ₂ H + NAD (P)

NH ₂

This reaction is catalyzed by the enzyme glutamate synthase (GUS). This combination of reactions consumes 1 molecule of ATP for the first stage and the equivalent of 3 MoIe-20 cool ATP for the second stage to produce 1 molecule of glutamate.

(Note: in the description means

the cofactor of the enzyme GDH or GUS in its oxidized form

the cofactor of the enzyme GDH or GUS in its reduced form, adenosine triphosphate, Adenosine diphosphate,

Deoxyribonucleic acid (this embodies the genetic material of the organisms concerned) and Pi phosphate ion J.

35 The exact mechanism by which ATP is consumed is not essential in the context of the description, but it does lead the greater consumption of ATP in the two-step mechanism

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25th NAD (P) NAD (P) H. ATP 30th ADP DNA

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overall for the conversion of a larger proportion of the starting methanol in carbon dioxide than in the direct mechanism.

With careful studies that the inventors

have carried out, it was found that the two-step mechanism in the production of single-cell protein by previously known processes despite the presence of a sufficient supply of free ammonia and / or ammonium ions in the reaction mixture predominates and that the more economical direct mechanism is possible if a genetically modified one Microorganism is used.

The invention relates to a process for the production of single-cell protein, characterized in that microorganisms, which contain genetic material that binds ammonia to the with the participation of the enzyme GDH route, breeds under aerobic conditions. The genetic material is e.g. B. plasmid DNA or plasmid hybrid DNA or phage DNA or phage hybrid DNA.

The process is carried out in an aqueous nutrient medium that contains methanol as the source of assimilable carbon and ammonia and / or ammonium ions in a concentration sufficient to maintain the GDH reaction mechanism, contains, and then single-cell protein or a mass containing the single-cell protein from the nutrient medium is won.

The process is suitably carried out under the conditions outlined in British Patent Specification 13,708,92 or 14 510 20 are known, but the total concentration should be in the zones of the maximum methanol concentration Ammonia and ammonium ion are preferably kept above 0.5 mmol / l will. As a result of the decreased ATP consumption, the conversion of methanol to carbon dioxide is lower overall, the process is therefore less exothermic and there is a

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less cooling is required to keep the temperature in the preferred range (34 ° C to 45 ° C). To the highest possible profitability To achieve, the active microorganism must predominantly and preferably exclusively on the methanol with the participation of the GDH enzyme.

According to the invention, microorganisms are one or more Species made available that are grown under aerobic conditions and are capable of being as a result of genetic Modification by plasmids or plasmid hybrids or phages or phage hybrids, each of which is a carrier of DNA, which prescribes the production of the enzyme GDH through the mechanism of ammonia binding that takes place with the participation of GDH Methanol with the formation of proteinaceous cell material

15 to convert.

Examples of the “basic organisms” from which the modified organisms are obtained are in particular the following microorganisms: Methylophilus methylotrophus (also known under the name Pseudomonas, methylotropha).

The properties of this kind are from the British patent specification 13 70 892 and cultures of strains of this species have been deposited under the following accession numbers: 25th

NCIB 10508 to 10515 and 10592 to 10596 (all inclusive ) _f

NRRL B5352 to B5364 (inclusive) and

FRI 1215 to 1227 (inclusive),

30 Pseudomonas methylonica

The properties of this kind are from the British patent specification 14 51 020 known, and a culture thereof has been deposited under the following accession numbers: 35

FRI 2247 (nov. Sp.) And 2248 (nov. Sp.)

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Other examples of basic organisms that can be used according to the invention can be used are:

Pseudomonas methanolica 5 GB-PS 13 527 28

ATCC 21704
Pseudomonas sp.

GB-PS 13 265 82 ATCC 21438 and 21439 10 Methylomonas methanolica GB-PS 14 202 64 NRRL B-5458
Pseudomonas utilis

GB-PS 14 440 72 15 FRI 1690 and 1691 Pseudomonas inaudita FRI 1693 and 1694

(NCIB = National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland;

NRRL = the collection maintained by the US Department of Agriculture, Peoria, Illinois;
ATCC = American Type Culture Collection; FRI = Fermentation Research Institute of Japan) 25

According to the invention, a method for production is also provided the genetically modified microorganisms by incorporating one or more plasmids or one or more phages, The carriers of the gene of the GDH mechanism are provided in the ways described above. When the modification is carried out via the plasmids, the procedure consists of the following steps:

(a) Creation of a mutant lacking genetic material that dictates glutamate synthesis via the GUS mechanism, from the basic organism;

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1 (b) Generation of a plasmid hybrid made from plasmid DNA is covalently linked to DNA that dictates the production of the enzyme GDH;

(c) introduction of the hybrid plasmid into the basic organism lacking the genetic material for the GUS mechanism;

(d) cultivation of the organism obtained under conditions which favor growth via the GDH mechanism and

(e) selecting one or more clones of organisms, that grow through the GDH mechanism.

If the modification is done via phages, then it exists Procedure consisting of the following steps:

(a) Creation (as described above) of a mutant lacking genetic material capable of glutamate synthesis prescribes the GÜS mechanism from the basic organism;

(b) Identification of a phage DNA or a temperate one Phages for the basic organism;

(c) Introduction of a piece of DNA, which prescribes the production of the enzyme GDH, into the phage or into the phage DNA, to make a phage hybrid;

(d) lysogenization of the basic organism lacking the genetic material for the GUS mechanism with the phage hybrid;

(e) culturing the organism obtained under conditions which favor growth via the GDH mechanism and

(f) Selecting one or more clones from organisms that grow via the GDH mechanism.

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Suitable general methods of making these modifications are found in British Patent 34 994/74 the applicant, to which the German Offenlegungsschrift 25 35 554 corresponds, is known. The description refers to this

5 patent is referred to.

The invention also provides new material masses in the form of the products, which in intermediate stages whichever way is used.

The mutant can be generated by standard methods, e.g. B. by treatment with a physical mutagen such as V, X-ray or UV radiation, with a chemical mutagen such as nitrous acid, a methanesulfonic acid ester (e.g. the Methyl ™ or ethyl ester), 5-bromuridine, 5-bromouracil, 2 -Aminopurine or nitrogen mustard or a biological mutagen such as B. a yu phage. Treatment which results in the formation of a deletion mutant is preferred. The mutant will normally remain capable of the GS response and therefore can be selected based on its ability to grow on glutamate but not on ammonia as the sole nitrogen source.

In addition to the fact that the mutant of the parent organism selected for further treatment lacks GUS, it also preferably lacks genes A and / or E ( trp ), which prescribe tryptophan synthesis. This is because a suitable way of generating the necessary plasmids or phage that prescribe the production of GDH also provides for the presence of DNA that prescribes trp , as described below.

The plasmids can be obtained from any source provided they are suitable for transfer into one of the parent organisms are capable. Examples of suitable sources are E. coli, Pseudomonas and Klebsieila. "P" incompatibility group plasmids are suitable because they are between a variety

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of gram-negative bacteria, which could be used as basic organisms, are freely transferable. E.g. this is Plasmid RP4 of the "P" group from E. coli can be transferred in this way and shows, wherever it is transferred, its special property (kta) of resistance to antibiotics Kanamycin, tetracycline and ampicillin.

The plasmid hybrid can be generated directly from many plasmid sources, including any plasmid sources present in the parent organism. However, because of the amount of experience gained in handling it, plasmid RP4 from drug-resistant E. coli is a particularly suitable starting point. The first step is then the creation of a derivative of EP4 which retains the ability to replicate in a wide variety of host organisms, but which does not contain the drug resistance marker. (The alternative method without the initial removal of the drug resistance marker is in principle possible, but poses a hazard to the environment when operated on a large scale.) This can be achieved by treating the genetic information on the RP4 with a class ii endonuclease that is, of the type that creates many DNA fragments with staggered breaks and thus "sticky ends", "shuffled" and then recombining the fragments in a random order. A suitable endonuclease is E Co R ₁ . The recombining of the fragments is effected enzymatically, suitably by ligase.

In a preferred form of this general method, the "shuffling" of the genetic information of the plasmid, whether or not the drug resistance tags are included, is carried out in the presence of an additional DNA fragment containing a genetic tag, e.g. B. trp genes. (Such genes can easily be "excised" as described below.

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from a suitable transducing phage genome using E Co R _1. ) The trp genes B, C and D from E. coli are "targets" for the restriction enzymes Hind III, which are therefore suitable for the subsequent introduction of heterologous DNA -Fragments can be used without destroying the selective labeling, because the genes A and E are not among the targets of Hind III and their functions can easily be selected. The plasmid fragments are then incubated with ligase, which reestablishes the covalent bonds in the double stranded DNA and creates some plasmids that have the trp genes but are not drug resistant.

Plasmids containing trp genes should be used especially when the parent organism contains a trp deficient mutation, as described above.

In any blending operation using E Co Bf ₁ , the conditions should be such that the E Co R ₁ * activity of this enzyme is provided, ie its activity at high pH and low Mg concentration. Under these conditions, the base sequence

25 .TTAA I IAATT *

recognized in the DNA double strand and cleaved to form the same "sticky ends" that the enzyme breaks at the Place

CTTAA JG

G IAATTC 35

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generated under normal conditions. The shorter recognition sequence of E Co R * means that the probability consists of many more fragments of any given double-stranded DNA molecule be split.

Note: A = adenine C = cytosine G = guanine 10 τ = thymine

In the phage method, the phage, if direct is used, belonging to the type, including mutants, which include the basic organism in the series of its natural hosts includes. If phage DNA is used, it can come from a natural phage or from a phage that is normally produced does not include the basic organism among its natural hosts.

Either way, if that's the manufacture gene prescribing GDH through step (c) of the plasmid pathway or through step (d) of the phage pathway in the GUS deficient mutant of the parent organism has been introduced using the presence of the GDH

25 organism recognized by its ability to use ammonia as its sole source of nitrogen.

The invention is illustrated by the tables below

explained in more detail: 30

Table 1 shows the simplest form of the plasmid pathway for generating the modified organism.

Table 2 shows a preferred form of the Tabelie Path 1,

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Table 3 shows a preferred modification of the route from Tables 2 and 4

Table 4 shows three forms of the phage method. 5

It is believed that these tables are generally straightforward to understand. With regard to Table 3, however, it should be explained that the DNA fragments B, which have been produced from a complete organism, contain the required GDH genes only in low concentrations and that consequently the probability that these genes will interact with plasmids P (kta , trp AE) link is not high. However, if the DNA fragments B are linked to DNA fragments C obtained from an A phage or from plasmids, the resulting DNA combination can be grown in a GDH deficient E. coli and then separated , and the product are GDH genes in a relatively high concentration, with only the DNA present in the phage or plasmids being present, which means an amount less than that present in a whole organism. The probability of linking with plasmids P (kta ~, trp AE) is advantageously increased in this way.

With the representatives listed below in the tables further explanations are necessary:

trp transducing λ phage: Well characterized material is available which contains genes AE of the trp operon.

DNA that prescribes the production of the enzyme GDH: This one DNA is identified by using a host using GDH 35

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generated, separates DNA therefrom, the DNA with a restriction enzyme such as. B. Hind III splits and with fragments of a Linked vector DNA made using the same restriction enzyme. With the linked DNA is then transfected into a GDH deficient host, which host becomes after transfection grown under conditions requiring the GDH pathway, and then the vector hybrid DNA is isolated therefrom.

It should be noted that where a specific enzyme is indicated in the tables, it is only one is an exemplary indication.

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Table 1

Basic organism

CIS GDH "

mutation

CO O CO CO

E. coli

insulation

GDH ^T

Basic organism

CIS
GDH "

Transfection

organism

P ~ group

plasmi & e

DNA ^F ragmente A

GDH +

DNA

Hind III

Hind III

organism

CIS

Ligase

-> P (GDH ⁺ )

DNA fragments B

VD

PO OO CO CD CD

Table 2

Basic organism

GUS ¹ GI) H "

mutation

(twice)

Basic organism

GUS_ GDH

tm A ~ E "

Transfection

. coli isolation

resistant

(kta ⁺ )

DNA ~ questionable duck

E Co R,

trp transducing lanbda phage

E Co R-,

tru A- E

Fragments

coli (GDH ⁺ )> DNA (GDH ⁺ )

Hind III

DNA fragments

Modifi "
more graceful

organism

CIS "
GDH ^4
4th

kta "

tm A - E

Ligase

co

kta

trp ⁺ A and

GDH ⁺

do

UD

Table 3

Lambda phage or plasmid

DNA

Hind III

E. coli (GDH)

DNA fragments B
or D

Ligase

Phage (GDH ⁺ )
PlasHid (GDH ⁺ )

DNA fragment P

formation of

DNA fragments A.

or
P (kta ~, trp ⁺ AE)

or just
trp A & E

Transfection,
Breeding,
insulation

Hind III

Hybrid phage (GDH) hybrid plasmid (GDH ⁺ )

cleaned

DNA

Table 4

Basic organism

Temperate phage

(Naturally)

GUS GDH

mutation

Basic organism

GTJS GDH

Addition of GDH ^T DNA

Hind III

DNA

Fragments lysogenization

Phage (GDH '
hybrid

Ligase

Addition of GDH ⁺ DNA

Hind III

- ^ fragments

Ligase

Phage
(not of course) Modified CIS

Organism ι GDH

DNA (GDH ⁺ )

CD

OO CO CD CD

Claims (12)

Claims 1. A process for the production of single-cell protein, characterized in that microorganisms which contain genetic material which prescribes the binding of ammonia on the path involving the GDH enzyme are cultivated under aerobic conditions. 2. The method according to claim 1, characterized in that the genetic material plasmid DNA, plasmid hybrid DNA, Is phage DNA or phage hybrid DNA. 3. The method according to claim 1 or 2, characterized in that it is carried out in an aqueous nutrient medium which Methanol as a source of assimilable carbon and ammonia and / or ammonium ions in a concentration sufficient to to maintain the GDH reaction mechanism, and that single-cell protein or a mass containing the single-cell protein is then obtained from the nutrient medium. 4. The method according to any one of the preceding claims, characterized in that the microorganisms by modification received one or more strains of the species Methylophilus methylotrophus. 5. Microorganisms of one or more species that are grown under aerobic conditions, characterized in that that they are able to, as a result of genetic modification, ^{XI / 17} 909813/078 qη ς ο - 2 - B 9194 fication by plasmids or plasmid hybrids or phages or phage hybrids, each of which is a carrier of DNA, the production of the enzyme GDH prescribes through the mechanism of ammonia binding which takes place with the participation of GDH Methanol for the formation of protein-containing cell material in a method according to one of the preceding claims utilize. 6. Microorganisms according to claim 5, characterized in that they by the genetic modification of one or several strains of the species Methylophilus methylotrophus were produced have been. 7 “Process for the production of genetically modified Microorganisms according to Claim 5 or 6, characterized in that the basic microorganisms are one plaanid or several plasmids , which are carriers of the gene of the GDH mechanism, incorporated, whereby one (a) creates a mutant from the basic microorganism that lacks genetic material that the synthesis of glutamate via prescribes the CIS mechanism, (b) creates a plasmid hybrid consisting of plasmid DNA, which is covalent with DNA that is responsible for making the enzyme GDH prescribes, is connected, (c) introduces the hybrid plasmid into the basic microorganism that is the genetic material for the GUS mechanism 30 is missing, (d) cultivating the resulting microorganism under conditions that favor growth via the GDH mechanism and (e) one or more clones of microorganisms from 909813/0787 - 3 - B 9194 1 that grow through the GDH mechanism. 8. Process for the production of genetically modified Microorganisms according to claim 5 or 6, characterized in that one or more phages, the carriers of the gene of the GDH mechanism are incorporated, whereby one (a) creates a mutant from the basic microorganism lacking genetic material that enables glutamate synthesis prescribes through the CIS mechanism, (b) a phage DNA or a temperate phage for the Basic microorganism identified, (c) introduces into the phage or the phage DNA a piece of DNA which prescribes the production of the enzyme GDH in order to create a phage hybrid, (d) the basic microorganism lacking the genetic material for the GUS mechanism with the phage hybrid lysogenized, (e) the obtained microorganism under conditions breeds that promote growth via the GDH mechanism and (f) selects one or more clones of microorganisms, that grow through the GDH mechanism. 9. The method according to claim 7 or 8, characterized in that the mutants of the basic microorganisms which are selected for further treatment, the genes A and / or E, which prescribe the tryptophan synthesis ( trp ), are missing. 909813/0787 - 4 - B 9194 10. The method according to claim 7, characterized in that the plasmid from E. coli, Pseudomonas or Klebsiella had received. 11. The method according to claim 10, characterized in that that the plasmid used is the RP4 plasmid obtained from drug-resistant E. coli. 12. The method according to claim 7, characterized in that that one can generate the plasmid hybrid in the presence of an additional DNA fragment which is a genetic marker contains, performs. 909813/0787