PROCEDURE FOR THE PRODUCTION OF VALUABLE MATERIAL FROM ORGANISMS

Procedure for the production of valuable material from organisms the invention concerns a procedure for the permanent production of valuable material in organisms, which have genetic information for the production of the valuable material and rule mechanisms for the controlling of production.

Rule mechanisms for the limitation of the bio-synthesis are on the one hand photograph transport systems and on the other hand metabolic bottlenecks. The photograph transport systems lead back the valuable material already produced again into the cell and give thus the signal for the bio-synthesis stop. In order to go around this mechanism, either these " natural " photograph transport systems are switched off or the gene necessary for the synthesis into a foreign organism without photograph transport systems or at least with a defective transport system brought, in foreign organisms are however perhaps metabolic bottlenecks present, which limit also the bio-synthesis.

By the term permanent production " is understood the continuous drain of the valuable material from the cells, whereby the cells produce new valuable material independently of their growth and increase due to the loss again. Permanent production can accomplish itself both in increasing and/or increasing and in not being awake and/or not increasing cells.

The biotechnological production of valuable material aims at it off, these valuable material by self-synthesis (and/or bio conversion from preliminary stages) to receive or by rekombinante Expression from metabolic pathways in production trunks in high concentration in the cells or in the medium. If the products are enriched in the cells, the state of the art for the production consists of unlocking the cells either to permeabilisieren or on other way for the delivery of the valuable material too to arrange (S. “bacteria milking”). The small final concentration of the valuable material in the product solution and the associated high costs of the product on cleaning are unfavorable (“downstream processing”).

The subject of the available invention is now a procedure, which makes the production possible of valuable material by the fact that hochaffine photograph systems for these valuable material are switched off or impaired in their function and/or production trunks are used, to which mentioned photograph systems are missing. The underlying surprising discovery is that the observed will keep concentration gradient over the cell membrane upright not alone thereby that the diaphragms are impermeabel for the valuable material, but also thus that hochaffine photograph systems in-transport " run out " valuable material rapidly again into the cell.

In this way an only apparently stationary situation develops, because in reality it concerns a constant Rezyklisieren of " drained " worth materials. The inventive idea consists of switching off or impairing these resumption systems, so that the produced valuable material the density gradient flow off following continuously into the surrounding medium. Due to the own regularization mechanisms the drained substances are supplemented. This recovery of lost valuable material is independent of growth and increase of the cells and cannot accomplish itself also with increasing and/or not increasing cells.

Under suitable conditions it acts around a conversion from substrate to the desired valuable material and corresponds themselves to the process of a bio catalysis. Depending on whether the new synthesis with the speed of the discharge step to hold knows or not, in the cells a concentration of the valuable material will adjust themselves, which corresponds to the natural concentration or is under it. In the equilibrium the bio-synthesis achievement corresponds to the product production rate. The procedure according to invention can be applied e.g. also to the production of osmotically induced valuable material under lowosmotic conditions (salt-poor media). The concentration maximally attainable in the medium corresponds (under the given conditions) maximally in the cell attainable to the concentration.

It can achieve molecular dimensions in the most favorable case with conventional fermentation techniques.

With view to as high a space-time yield as possible the procedure according to invention is supplemented by suitable measures, which support the self-bio-synthesis of the valuable material and/or eliminate ( in case of the transmission of a metabolic pathway on an appropriate producer trunk) possible metabolic bottlenecks. This e.g. is . to realize by the fact that Medien-und growth conditions are combined vary will and/or the transferred metabolic pathways with further genes (aide genes), which eliminate regulatorische or functional bottlenecks.

Valuable material, e.g . with the procedure to be won can, are natural-prove highly concentrated stress protective agents, so-called compatible Solute, in addition, valuable material in small zytoplasmatischer concentration, if they membranpermeabel are and the density gradient is sufficient to the surrounding medium, in order to cause EFF lux from the cells. In the first case it preferably acts around the gene products of so-called extremophiler organisms like e.g. halophile or osmotolerante Eubakterien (Bacteria) and/or.

Archaebakterien (Archaea), halophile or osmophile eukaryontische organisms (yeasts or algae inclusive macro algae), thermophile Eubakterien (Bacteria) and/or Archaebakterien (Archaea) and coolingloving microorganisms. In the second case the whole spectrum of possible macromolecular and low-molecular substances is addressed, which natural-proves either or can by genetic " engineering " on the way of the bio-synthesis and/or bio conversion by cells be produced. If the bio-synthesis of the valuable material does not take place automatically, it can e.g. by stress factors like e.g.

Heat, extreme cold weather or osmotic imbalance or however by other factors to be induced, those in molecular biology are common (e.g.

IPTG) Exemplarily following substances and material classes are mentioned: all Primär-und Sekundärmetabolite, which can be synthesized by cells, z.

B. Amino acids, Peptide, proteins, organic acids, sugars and derivatives, fatty acids, nucleotides, antibiotics, vitamins etc., among them in high concentration in particular non-reducing sugars (e.g. Trehalose, Mannosylglycerat, Mannoslyglyceramid), Polyole and derivatives (e.g. Sorbitol, Cyclite, Diinositphosphat, cyclisches Diphosphoglycerat, Diglycerinphosphat) as well as zwitterionische amino acids and derivatives (e.g. Prolin, betaines, Ectoine, N-acetylated and/or N-carbomoylierte Diaminosäuren or amino acid amides and Peptide).

As exemplary products, which can be won in procedure according to invention, the compatible Solute from the group of the Ectoine is to be called , either as pure substance or in combination with other materials of bio molecules (nucleic acids, proteins, Lipide), complex '' self assembly " structures (e.g. Ribosomen; Protein DNA, Protein-Protein-und protein of sugar complexes; Lipidfilme, diaphragms etc.) or also whole cells stabilsieren [there Costa et al. 1997]. Further application possibilities lie in the use than water-binding substance (more moisturizer) and active substance in Hautund toiletries, than Probiotika, Nutraceuticals and in pharmaceutical formulations.

The elimination and/or influence according to invention from transport systems to the purpose of the production of valuable material can take place in different way: a) by mutation of the coding genes in the producing organisms b) by inhibition/lnaktivierung the Transportsytems on protein level c) by production of the genes for a certain bio-synthesis way and transmission to a production trunk, either does not natural-prove appropriate transport systems has or with this were switched off the genes, which code for the specific valuable material transporters, can with the help of different genetic methods be mutated, e.g. Transposonmutagenese or purposeful mutation (e.g. Deletionen) by PCR techniques.

An organism should be selected as production trunk for the production of valuable material, which if possible few Tranporter for the Sub which can be produced punch possesses. Such a genetic background has the advantage the fact that possibly only one transporter must be switched off and thus a production trunk is designed, which grants rearing conditions the valuable material to that under none again to take up and can metabolisieren. This extends the rearing possibilities and production engineering of the inventive procedure.

Alternatively an impairment of the transport systems is possible thereby that Antimetabolite are added to the medium, which either to the transporter bind (without to be transported) and thus or in other way a change transport haven in to cause, so that it no more or only in reduced mass in the situation is, to take over the function of valuable material transport.

The transmission of genes for valuable material production is operated, around well-known producer trunks like e.g. E. coli to be able to use Bacillus subtilis and Cotyne bacterium glutamicum to become in this way independent and from the natural producer trunks to which can be cultivated partially very badly or unusual rearing conditions to require.

In case of the Ectoinproduktion a transmission of the genes coding for the synthesis enzymes of the Ectoin becomes on not halophile producer trunks (like e.g. E. coli) desired, in order to solve Korrosions-und of disposal problems justified in the use of salzhaltiger media. During the genetic transmission of a whole metabolic pathway however the complex regularization in the receiver organism is to be considered. So the Ectoingene can not halophilen also in organisms to be exprimiert [Galinski & Louis 1997, Louis & Galinski 1997], which are production rate in addition, limits there osmotically adjusted and by metabolic bottlenecks (e.g. on the stage of the Aspartatkinase).

For the execution of the procedure according to invention cultivation is favourable in a fermenter. The boundary conditions are to be selected in such a way that the culture as for a long time as possible in the growth phase stay and/or are reached high cell densities (e.g. Hochzelidichte fermentation) or the cells by nutrient limitation from growth to be prevented, so that the ange botene C-source is converted as completely as possible in valuable material. In case of the transmission of a bio-synthesis way for the valuable material on a suitable producer trunk additionally suitable conditions must be created, in order to induce the genes (if necessary in combination with aide genes). The descriptive production strategy could be modified also such that cells by immobilization or by a diaphragm (e.g.

Dialysis hose) from the production solution separate are. After reaching the maximum product concentration the cell mass could be transferred into new medium (“tea bag principle”) and so the process to be continuously continued. The described procedure presents itself in particular if the valuable material is formed over an energy-independent synthesis way (e.g. the amino acid derivative Ectoin). In this case it is possible to convert the offered C-source completely into valuable material (“bio conversion”), so that the organism takes over the function of a biocatalyst.

If an optimal production trunk is selected and/or designed, it is possible with the descriptive production engineering to enrich the desired valuable material in high concentration (up to 0.5 M and more) outside of the cell without the cells from the medium abgeerntet and unlocked, temporarily permeabilisiert or in other way (e.g. Dilution stress) to the delivery of the substances to be arranged would have. The production cells remain to ideal way so long in the medium to the medium components (e.g. C source) are completely used up, in order to simplify the following Aufreinigung.

If the valuable material should flow off only insufficiently over the diaphragm, would leave itself the procedure for e.g. osmophile or halophile organisms modifzieren also in such a way that the cells are exposed to a dilution stress. This osmotic " down-squat " induces the delivery of the valuable material into the medium. In relation to the conventional " bacteria milking " the procedure described here possesses the advantage that production cells and product do not have to be separated according to the product delivery no more, since the cells lost the ability to take up the valuable material again. Rather directly the concentration at solved materials following the osmotic dilution stress (z can.

B. NaCI, PEG etc.) to be again increased (“upshock”) and renewed production by valuable material to be introduced. Similarly as the classical " bacteria milking " also this process can be arranged continuous.

The physiological principle, which is the basis the invention and it made possible to win by change and/or impairment of Solutetransportern of valuable material is to be explained on the basis the TRAP transporter (TRAP-T) with the name TeaABC from Halomonas elongata.

TRAP T systems are transporters, which refer their energy for the transport of materials from the Membranpotentiaf of the cell and from there secondary transporters are called. TRAP-T of systems have that a substrate connection protein, outside of the cell are located and a diaphragm domain.

The diaphragm domain of the TRAP-T Syteme consists either of a small and a large Transmembranprotein or a fusion protein of the two diaphragm proteins. The transporter the TeaABC from H. elongata is a TRAP-T system with two diaphragm protein. TeaABC transports the compatible Solute Ectoin and Hydroxyectoin (valuable material) with osmotic stress into the cell inside (Cytoplasma) and makes the enrichment possible of these substances in high molecular concentration, in order to manufacture an osmotic equilibrium between the cell and the outer medium. TeaABC is activated by osmotic stress, this concerns a osmoreguliertes transport system. The major task of TeaABC does not exist to take up now however to it cellstrange Ectoin from the medium.

This is explained on the one hand by the fact that Ectoin in the medium does not natural-prove in high concentration occurs. In addition it showed up that the destruction of the transporter leads to a continuous loss of Ectoin. Obviously TeaABC is used mainly to transport cellown Ectoin again into the cell back. The loss of Ectoin is now however no nonspecific procedure. Rather it seems in such a way that the Solut leaves the cell over a special pore, if sufficiently Ectoin is in the ZeNinneren present by Eigensysnthese. Outside of the cell (Periplasma) concerned, Ectoin is bound by the Substrabindeprotein TeaA of the transporter TeaABC and transported again into the cell.

The transport of Ectoin back into the cell serves as signal that sufficiently compatible Solut Ectoin was synthesized and the Ectoinsysnthese of the cell thereupon adjusted. As a mediator between the active transporter and the synthesis of Ectoin a modulator protein with the name serves TeaD. TeaD is coded by a gene, which lies probably beside the genes of the transporter TeaABC and together with these one exprimiert. TeaD must interact and recognize with the transporter TeaABC that the transport system takes up Ectoin. This activity is passed on probably by Phosphrylierung and leads to the appropriate negative regularization of the Ectoinsynthese. The meaning of TeaD as modulator was confirmed by mutation experiments.

The destruction of TeaD leads active transporter with TeaABC to the fact that Ectoin from the cell flows. Although TeaABC Ectoin transports, this signal cannot be passed on and the synthesis of Ectoin stays on to high level. A destruction and/or an impairment of TeaABC leads thus not only to it that Ectoin leaves the cell, but also to the fact that the loss becomes balanced by increased synthesis, since the cell does not receive a negative signal by TeaABC and TeaD. Mutanten, as they are represented in procedure example 1, are from there both Ausscheidermutanten and over producers for valuable material.

The goal of winning valuable material by means of changed and/or impaired transport systems can be achieved also by the fact that the bio-synthesis genes for the desired valuable material in producer trunks with according to changed/impaired or completely which are missing transport systems is exprimiert. The advantage consists of the fact that only once a suitable producer trunk (e.g. an E. coli trunk, to which all osmoregulierten transport systems are missing) must be produced, in which then different valuable material can be produced.

While the rekombinante Expression is in principle controlled by individual foreign genes in producer trunks, the problem however always develops during the transmission of whole metabolic pathways that also aspects of regularization of the enzymatic cascade (e.g. the regularizations of the cytoplasmatischen concentration of preliminary stages) must be included also. For the case of the transmission of the gene cluster for the Ectoinbiosynthese this means that the biosynthetic bottleneck (a strictly adjusted Aspartokinase) for this bio-synthesis way (“bottle neck”) opens a deregulierte Aspartokinase, ge only by bringing in a further gene, i.e. become can.

Procedure example 1 production of the valuable material Ectoin from the Eubakterium Halomonas elonqata DSM IT by mutation of the Ectointransporters TeaABC (A) construction of a Ectoin Transportmutante of Halomonas elongata the gene teaC, which codes TeaABC from TRAP T family for the large Transmembranuntereinheit of the osmoregulierten Ectoin transporter [forward et al. 1997], are deletiert with the help of the SOE PCR technology (splicing by overlap extension). The resulting DNA fragment with now connected the sequences upstream and downward of feaC becomes with the help of the konjugativen Plasmids pK18mobsacB [shepherd et al.] brought into the type of game by H. elongata.

Via homologous recombination an exchange the type of game DNA takes place against those of the Deletionsfragmentes. The Dele tionsmutante H coming out from it. elongata KB2 (AteaC) is not any more able to take up Ectoine with osmotic stress.

(B) Culture medium glucose mineral salt medium (WM) : NaCI (100 g/l) ; KCI (1 g/l) ; MgS04x7H20 (6.5 g/l) ; NH4C1 (3 g/l) ; K2HPO4 (0, 5 g/l) ; CaCI2x2H20 (0, 01); FeS04x7H20 (0,01); D-GlucosexH20 (11 g/l), yeast excerpt (0.1 g/l), trace element solution (TES; Claus's et al. 1983) (1 mull) ;

Vitaminlösung VA (Imhoff & Trüper 1977) (1 ml/l) (C) cultural conditions H. elongata KB2 (AteaC) in a bio act a V-fermenter with an active volume by 4 I with 30 C in a fed batch process is tightened. Glucose concentration in the cultural solution becomes by addition of a concentrated fed batch solution (594.6 g/1 glucose mono hydrate; 133.6 g/1NH4CI ; 87 g/L K2HP04 ; 100 g/L NaCI) between 5 g/L and 15 g/L held.

The culture ventilated with compressed air (3 I/min), the agitator number of revolutions amounts to 250 rpm. The regularization of the pH of value on pH 7.2 is made via the sterile addition from HCI (1 M) or NaOH (5 M) by hose pumps. The increase of the cell number is pursued by determination of the cell protein content and the measurement of the optical density with 600 Nm (OD600 ).

(D), From analytics the samples for the determination of the glucose content in the medium, the cell protein content, the OD600 and the Ectoingehalts in the medium over a Kanüle in the fermenter cover be removed. For the determination of the Ectoingehalts in the medium the cells are abzentrifugiert and the medium projections on a isokratischen HPLC with UV detection are measured.

(F) enrichment of Ectoin in the medium Ectoin (however no Hydroxyectoin) enriches itself in the process of the entire fermentation process in the medium on (figure 1). With the reaching of the maximum OD600 of 54,0 the organism loses approx. 8.2 g Ectoin. Thus is approx.

60% of the entire Ectoins in the medium. Also in the stationary phase H synthesizes. elongata KB2 Ectoin, and the final concentration at external Ectoin amounts to 15 gel (approx. 100 mm). Thus approx. 90% of the formed Ectoins are into the medium dismiss. With consumption of the C-source glucose H.elongata stops the production of Ectoin.

Procedure example 2 production of the valuable material Ectoin by construction of a vector, which combines the Ectoinsvnthesegene with an aide gene ( Asparatkinase derequlierte ). for transmission to transport-defective production trunks by the disclosure writing DE 199 25 615 A1 (7.12.2000) a procedure is revealed, which is suitable, to the complete Ectoinbiosyntheseweg under simultaneous opening of possible metabolic bottlenecks on other organisms (e.g. E. coli DH5a) to transfer and Ectoin in this way into salt-poor media to produce.

Against expectations thereby however no overproduction was obtained, probably, because a suitable promoter was missing to the Ectoingenen and because on protein level insists a feedback regularization of the Ectoin genes. This procedure example describes the construction of a vector pAKECT1, in which the Ectoin genes stand under control of the own promoter (ectUP ) and are combined with the aide gene IysC ( from Corynebacterium glutamicum MH20-22B). In this way a Ectoin bio-synthesis can be obtained under opening by metabolic bottlenecks in E. coli, which leads in transport-defective producer trunks to the desired overproduction in an environment of small Salinität.

(A) Construction of the vector pAKECT1 a nucleic acid fragment from that Gram positive, halophilen bacterium Marino coccus halophilus, which carries the genes for the synthesis of the compatible Solutes Ectoin (ectA, ectB, ectC), and a nucleic acid fragment from that for Gram positive bacterium Corynebacterium glutamicum MH20-22B [Cremer et al. 1991], which carries the gene for an enzyme Aspartokinase resistant to " feedback " inhibition (IysC), became by means of molecular-biological standard techniques from the Plasmiden pOSM12 and/or pRK1 ligiert into the vector pHSG575.

Contrary to Plasmid pAKECT2, with which the Ectoin Gencluster under control of the lac promoter and the gene stand for the Aspartatkinase under control of the TAC promoter, the Ectoin genes in Plasmid pAKECT1 are controlled by the own modulator sequences ectUP. Figure 2. gives an overview of the individual Klonierungsschritte for the construction of the Plasmids pAKECT1.

(B) Transmission of the vector on E. coli DH5a by CaCl2-Transformation [Hanahan 1983] was transferred the Konstrukt into Escherichia coli to DH5a. The identification of the clones with strange DNA took place on genetic way by means of Plasmidisolierung, restriction-digests and Agarose Gelelektrophorese according to well-known molecular-biological methods [Ausubel et al. 1992].

(C) Determination of the intrazellulären Ectoingehalts in E. coli DH5the rekombinante Plasmid containing receiver organism Escherichia coli DH5a became in glucose mineral salt medium MM63 after Larsen et al .

[1987] with different salt concentrations (1 5% NaCI) cultivates. The rearing took place in 250-ml Erlenmeyer flask with chicanery in 100 ml medium (rotation shaker Innova 4232 of the companies

New Brunswick, Edison; 150 RPM, 37 C). The won cell material was dried by means of Zentrifugation harvested (4 C , 2,800 g, 15 min, centrifuge universal R16, Hettich , Tuttlingen) , with 70 C and extracted for the production intrazellulär of the available compatible Solute. The analysis of the intrazellulären Solutespektrums took place by means of isokratischer HPLC [Galinski 1986]. The Ectoinproduktion of AKECT2-und AKECT1-Konstrukten became in each case to compare, without and with IPTG induction (1 mm).

Figure 3 describes the intrazellulären Ectoingehalt as a function of the Salinität of the medium (in mmol/g dry weight). It is clearly recognizable that by the IPTG induction of the aide gene IysC in pAKECT1 the metabolic bottleneck are repaired and clearly higher Ectoinmengen is obtained.

Surprisingly the Ectoinkonzentrationen obtained with pAKECT1 is almost identical to those, which are obtained by the control trunk pHSG575, if Ectoin in the medium is offered. To it becomes clear that the transport system for Ectoin (and not the bio-synthesis achievement of the cells) determines the internal concentration. There the maximum concentration with 2% NaC! already almost, the goal of a rekombinanten Ectoin Zellfabrik for salt-poor media is achieved by the fact is realised that the vector will transfer pAKECT1 according to procedure according to invention to receiver organisms, to which the transport systems for Ectoin are missing or whose transport systems are impaired switched off or in their function.

LITERATURE Ausubel FM, Brent R, Kingston RH, moorlands DD, Seidman JG, Smith, Struhl K (eds.) (1992) Short protocols in Molecular biology, 2 " D edition. John Wiley & Sons, New York.

Claus's D., Fahmy F., Rolf H.J., Tosunoglu N. (1983) Sporosarcina halophila frame Nov., to obligatory, slightly halophilic bacterium from march salt soils. Syst Appl Microbiol 4: 496-506; Cremer J, Eggeling L, Sahm H (1991) control OF the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression OF the individually corresponding gene. Appl Environ Microbiol 57: there 1746-1752 Costa ms, Santos H, Galinski I/O (1998) on overview OF the role and diversity OF compatible solutes in Bacteria and Archaea. Automatic data processing Biochem closely Biotechnol 61: 117-153 forward, J.A., Behrendt M.C, Wyborn N.R., CROSS R., and Kelly D.J.

(1997) TRAP transporter: A new Family OF Periplasmic Solute transport of system Encoded ny the dctPQM gene OF Rhodobacter capsulatus and by Homolgs into various Gram negatives Bacteria. Journal OF Bacteriology 179: 5482-5493; Galinski I/O (1986) salt adaptation by compatible Solute with halophilen phototrophen bacteria. Thesis, University of Bonn.

Galinski I/O, of Louis P (1997) procedure for the transmission of the ability for the bio-synthesis kompatiblerSolute and the associated stress tolerance on organisms. OS DE 196 10 972 A1 Hanahan D (1983) Studies on transformation OF Escherichia coli with Plasmids. Mol of Biol 166: 557-580. Imhoff J. F., Trüper H.G. (1977) Ectothiorhodospira halochloris frame Nov., A new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch Microbiol 114: 115-121.

Larsen pi, Sydnes process card, Landfald B, river ACRE (1987) Osmoregulation in Escherichia coli by accumulation OF organic osmolytes : betaines, glutamic acid and trehalose. Arch Microbiol 147: 1-7 of Louis P, Galinski I/O (1997) Characterization OF of gene for the biosynthesis OF the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143: 1141-1149 shepherds, A., A. dip, W. hunter, J. Kalinowski, G. Thierbach, and A. Pühler.

(1994) Small mobilizable multi-PUR-float cloning vectors derived from the Escherichia coli plasmids pK18 and pK19 : selection OF defined deletions into the chromosome OF Corynebacterium glutamicum. Genes 145: 69-73