DE102021124566A1 - PROCESS FOR THE RECOMBINANT PRODUCTION OF POLYPEPTIDES - Google Patents

Abstract

Summary The present invention relates to methods for the recombinant production of polypeptides from thermophilic and hyperthermophilic organisms in mesophilic host organisms and their use for the production of extremolytes.

Description

field of invention

The present invention relates to methods for the recombinant production of polypeptides from thermophilic and hyperthermophilic organisms in mesophilic host organisms and their use for the production of extremolytes.

background

Extremolytes are stress protection molecules that are formed by extremophilic microorganisms and protect them from the extreme environmental conditions of their respective habitat, in particular high salt concentrations and extreme temperatures. For this purpose, these molecules are formed or taken up in comparatively high concentrations within the cell and protect sensitive cell structures such as membranes, proteins and nucleic acids from damage and/or degradation. Although highly diverse, most fall into two chemical categories: a) amino acids and their derivatives such as alpha and beta glutamate, glycine betaine, ectoine, hydroxyectoine, and b) saccharides, polyols and their derivatives such as trehalose , mannosyl glycerate and glycerol. The exceptional protective properties of these molecules make them interesting additives in a wide variety of commercial applications, such as food, healthcare and cosmetics. In particular, the extremolytes ectoine and hydroxyectoine, which are formed by halophilic bacteria, in particular Halomonas elongata, are already being used commercially. Extremolytes are also known for other classes of microorganisms, such as hyperthermophilic bacteria. These are often heterosides such as glucosylglycerol, glucosylglycerate, mannosylglycerate and mannosylglyceramide, and phosphorylated compounds such as di-myo-1,1'-inositol phosphate, alpha-diglycerol phosphate and cyclic 2,3-diphosphoglycerate. So far, however, these have not been studied much in terms of their potential for biotechnological and industrial applications. This is particularly due to the fact that no economically viable manufacturing processes are known to date. For example, the extremolyte cyclic 2,3-diphosphoglycerate (cDPG), found in hyperthermophilic methanogens (archaea) such as Methanothermus fervidus, Methanpyrus kandleri and Methanothermobacter thermoautotrophicus at concentrations of 0.3 to 1.1 M, is known to plays a role in the thermoprotection of proteins and increased temperature stability has already been reported for a number of enzymes (Shima, S., et al., Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri. Arch Microbiol, 1998. 170(6): pp. 469-72; Hensel, R. and I. Jakob, Stability of Glyceraldehyde-3-Phosphate Dehydrogenases from Hyperthermophilic Archaea at High Temperature. Systematic and Applied Microbiology, 1993. 16(4 ): pp. 742-745; Borges, N., et al., Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes.Extremophiles : life un der extreme conditions, 2002. 6(3): pp. 209-216). It has also been reported that it can protect plasmid DNA against oxidative damage by hydroxyl radicals and also act as a superoxide scavenger (Lentzen, G. and T. Schwarz, Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol, 2006. 72 (4): pp. 623-634). Despite the interesting properties of cDPG, no efficient manufacturing processes are known to date. One of the reasons for this is that low cell yields and difficult cultivation conditions hinder a cell-based in vivo process.

In general, there is a need for alternative manufacturing processes for extremolytes. Alternative manufacturing processes are possible to meet this need, with enzymatic processes being of particular interest here. In a first step, however, such enzymatic methods require the provision of key enzymes which are required for the synthesis of the respective extremolyte. It can be provided by recombinant expression in a suitable host organism. The present invention fulfills this need and provides such methods of expression of polypeptides from thermophilic and hyperthermophilic organisms in mesophilic host organisms and the use of polypeptides obtainable therewith for the production of extremolytes, such as in particular cDPG.

Summary of the Invention

The present invention is based on the finding of the inventors that a key enzyme for the production of cDPG, namely the cyclic 2,3-diphosphoglycerate synthetase (cDPGS) can be produced and stabilized in sufficient quantities by heterologous expression in order to achieve an efficient one-step in vitro enable synthesis. This finding can be generalized to other polypeptides from thermophilic and hyperthermophilic organisms.

1. a) Einbringen eines Nukleinsäuremoleküls, welches für das Polypeptid aus einem thermophilen oder hyperthermophilen Organismus kodiert, in den mesophilen Wirtsorganismus;
2. b) Kultivieren des mesophilen Wirtsorganismus unter Bedingungen, die die Expression des Polypeptides erlauben; und
3. c) Isolierung des Polypeptids aus dem mesophilen Wirtsorganismus.

In a first aspect, the invention therefore relates to a method for the recombinant production of a polypeptide from a thermophilic or hyperthermophilic organism in mesophilic host organisms, comprising

1. a) introducing a nucleic acid molecule which encodes the polypeptide from a thermophilic or hyperthermophilic organism into the mesophilic host organism;
2. b) culturing the mesophilic host organism under conditions allowing expression of the polypeptide; and
3. c) isolation of the polypeptide from the mesophilic host organism.

In various embodiments, the nucleic acid molecule encoding the polypeptide from a thermophilic or hyperthermophilic organism is codon-optimized for expression in the mesophilic host organism.

The cultivation of the host organism in step b) of the process can be carried out under mesophilic conditions, in particular in a temperature range between 10 and 45°C.

1. a) Lyse des Wirtsorganismus;
2. b) Erhitzen des Lysats aus Schritt a) oder eines Überstands, welcher aus dem Lysat erhalten wird, auf eine Temperatur von 50 bis 95°C, vorzugsweise 60 bis 90°C, zur Präzipitation der wirtseigenen Proteine und Polypeptide; und
3. c) Isolieren des löslichen Überstands von dem in Schritt b) erhaltenen Präzipitat.

In various embodiments of the method, the step of isolating the polypeptide from the mesophilic host organism comprises:

1. a) lysis of the host organism;
2. b) heating the lysate from step a) or a supernatant obtained from the lysate to a temperature of 50 to 95°C, preferably 60 to 90°C, to precipitate the host proteins and polypeptides; and
3. c) isolating the soluble supernatant from the precipitate obtained in step b).

These isolation steps a) to c) can be followed by one or more additional steps which serve to further purify or isolate the polypeptide from the solvate obtained in step c). Such methods include, for example, conventional size exclusion chromatography methods.

In the methods of the invention, in various embodiments, the polypeptide may be devoid of an affinity tag, preferably a non-native peptide sequence.

In various embodiments, the polypeptide is an enzyme. Such enzymes can be selected from glycosyltransferases (EC class 2), hydrolases (EC class 3), lyases (EC class 4) and ligases (EC class 6). Particularly suitable enzymes are esterases, lipases, glycoside hydrolases, dehydratases, aldolases, decarboxylases and synthetases.

In various embodiments, the polypeptide is an enzyme that catalyzes at least one step in the production of an extremolyte.

In various embodiments, the enzyme is selected from trehalose glycosyl-transferring synthase (TreT) and cyclic 2,3-diphosphoglycerate synthetase (cDPGS), in particular the cDPGS from Methanothermus fervidus and functional variants or fragments thereof.

In the methods of the invention, the thermophilic or hyperthermophilic organism from which the polypeptide to be expressed is derived may be a prokaryote, for example selected from bacteria and archaea, in particular from bacteria of the orders Thermales, Thermotogales and Aquiificales or Archaea of the lines Crenarchaeota and Euryarchaeota. In the case of the Archaea of the lines Crenarchaeota and Euryarchaeota, organisms of the classes Thermoprtei, Desulfurococcales, Sulfolobales, Methanopyri, Methanococci, Methanobacteria, Thermococci, Thermoplasmata and Archaeoglobi are particularly suitable.

In a further aspect, the invention relates to a method for the enzymatic production of extremolytes, the method comprising the use of recombinantly produced enzymes from thermophilic or hyperthermophilic organisms which can be obtained according to one of the methods of the invention.

In various embodiments of such methods, the extremolyte is cyclic 2,3-diphosphoglycerate (cDPG) and the polypeptide is cyclic 2,3-diphosphoglycerate synthetase (cDPGS).

In yet another aspect, the invention relates to the use of the polypeptides produced by one of the methods of the invention for the production of extremolytes, in particular for the in vitro production of extremolytes.

In a further aspect, the invention relates to a method for producing cyclic 2,3-diphosphoglycerate from 2,3-diphosphoglycerate (2,3-DPG) using a recombinantly produced cyclic 2,3-diphosphoglycerate synthetase (cDPGS). In various embodiments of the invention, the method is an in vitro method in which a recombinantly produced and isolated cyclic 2,3-diphosphoglycerate synthetase (cDPGS) is used. The process is preferably in one step.

In various embodiments, the enzyme cDPGS is the cyclic 2,3-diphosphoglycerate synthetase (cDPGS) from Methanothermus fervidus or a functional variant or fragment thereof. In different Embodiments, the enzyme comprises the amino acid sequence according to SEQ ID NO:1 or a functional variant thereof which has at least 80% sequence identity to the sequence given in SEQ ID NO:1 over the entire length. The enzyme can also consist of the sequences mentioned. The enzyme is obtainable according to the invention by the methods described herein. Also suitable are homologues of the cDPGS from Methanothermus fervidus from other organisms, such as Pyrococcus woeseii. In various embodiments of the invention, a cDPGS from a mesophilic organism, such as Methanobrevibacter and Rubrobacter species, can also be used in this aspect of the invention. Such homologues can have sequence identities of 40% or more to the amino acid sequence given in SEQ ID NO:1.

In various embodiments of the invention, the recombinant enzyme is produced by heterologous expression in a host organism, in particular Escherichia coli. For this purpose, a nucleic acid molecule which encodes the desired cyclic 2,3-diphosphoglycerate synthetase (cDPGS) is introduced into the host organism and expressed there under suitable conditions. In various embodiments, this nucleic acid molecule comprises a nucleotide sequence which encodes the amino acid sequence according to SEQ ID NO:1 or a functional variant thereof which has at least 80% sequence identity to the sequence given in SEQ ID NO:1 over the entire length. This nucleotide sequence can be codon-optimized for expression in the desired host organism and, in various embodiments, can comprise or consist of the sequence according to SEQ ID NO:2 or SEQ ID NO:3.

character list

• : cDPG synthesis. Conversion of 2-phosphoglycerate (2PG) to 2,3-diphosphoglycerate (2,3DPG) by 2-phosphoglycerate kinase (2PGK) and formation of cyclic 2,3-diphosphoglycerate (cDPG) by cyclic 2,3-diphosphoglycerate synthetase ( cDPGS). Both catalytic steps are ATP-dependent and additionally require magnesium ions.
• : List of primers (SEQ ID NOs:4 - 9) for the cloning of M. fervidus cdpgs (A) and nucleotide sequences of the cdpgs gene (SEQ ID NO:2) and of the codon-optimized cdpgs gene (SEQ ID NO:3) for expression in E. coli (B).
• : Size Exclusion Chromatography (SEC) purification chromatogram. The purified soluble fraction of cDPGS after heat precipitation at 75°C (5.6 mg) was loaded onto a Superdex200 size exclusion column. The box encloses the peak containing the recombinant cDPGS. Fractions containing pure enzyme were pooled and concentrated.
• : Expression and purification of recombinant cDPGS from M. fervidus heterologously expressed in E. coli. The cdpgs gene was cloned into pET24a with C-terminal His-tag and expressed in E. coli BL21(DE3) codon plus. The protein fractions (5-10 µg) were analyzed by SDS-PAGE (12.5%) after successive purification steps and stained with Coomassie Brilliant Blue. Mbn: membrane fraction, CE: crude extract, CL: crude lysate, HP: soluble fraction after heat precipitation (75°C, 30 min), FT: flow-through fraction-, W: wash fraction-, E1: elution fraction 1 - from Ni-TED affinity chromatography , M: protein markers, prestained protein ladder (Thermo Fisher Scientific, Carlsbad, USA). Large amounts of the recombinant protein were detected in the insoluble membrane fraction, marked by the box.
• : Expression and purification of recombinant codon-optimized cDPGS from M. fervidus and immunological detection using an anti-His-tag antibody. The codon-optimized cdpgs gene was cloned into pET15b with N-terminal His-tag and expressed in E. coli BL21(DE3)-codon-plus. After successive purification steps, the protein fractions (5-10 µg) were analyzed by SDS-PAGE (12.5%) (A) and stained with Coomassie Brilliant Blue. An anti-His-tag antibody (Thermo Fisher Scientific, Carlsbad, USA) was used for the immunological detection of His-tag cDPGS (B). Mbn: membrane fraction, CE: crude extract, CL: crude lysate, HP: soluble fraction after heat precipitation (75°C, 30 min), FT: flow-through fraction-, W: wash fraction-, E1-2: elution fraction 1-2 - from the Ni -TED affinity chromatography, M: protein markers, (A) unstained and (B) prestained protein ladder (Thermo Fisher Scientific, Carlsbad, USA).
• : Purification of recombinant 2,3-diphosphoglycerate cyclic synthetase (cDPGS) from 1.7 g E. coli cells.
• : Purification of recombinant cDPGS from M. fervidus heterologously expressed in E. coli. The codon-optimized cdpgs was cloned into pET15b without an affinity tag. Expression was optimized in E. coli BL21(DE3) codon plus and the protein was purified by heat treatment (HP) and size exclusion chromatography (SEC). Protein fractions (5-10 µg) were added after successive purification steps analyzed by SDS-PAGE (12.5%) and stained with Coomassie Brilliant Blue. Mbn: membrane fraction, CE: crude extract, HP: soluble fraction after heat precipitation (75°C, 30 min), SEC: fraction after size exclusion chromatography, M: protein markers, unstained protein ladder (Thermo Fisher Scientific, Carlsbad, USA).
• : Influence of salt and pH on the activity of the recombinant cDPGS. Influence of KCl (0-500 mM) (A) and pH (5.5-8) (B) on the activity of partially purified cDPGS (16.8 µg) after heat treatment (75°C, 30 min). The formation of ADP from ATP was coupled to the oxidation of NADH by rabbit muscle pyruvate kinase (PK) and lactate dehydrogenase (L-LDH). Activity was determined as a decrease in absorbance at 340 nm at 55°C. The pH optimum was determined in the buffer system described in the example section (material and methods). 400 mM KCl and 2.5 mM MgCl ₂ were added to the associated buffers. 100% of the relative activity corresponds to a specific activity of 1.8 U mg ^-1 .
• : Enzymatic properties of the recombinant cDPGS. The enzymatic activity (A & B) was measured at 55°C (340 nm) using 3.7 µg of purified enzyme by coupling the formation of ADP from ATP with the oxidation of NADH with pyruvate kinase (PK) and lactate dehydrogenase ( L-LDH) from rabbit muscle as auxiliary enzymes. The specific activity of the purified cDPGS was determined with 2,3-DPG concentrations of 0-8 mM and 2.5 mM ATP (A) and ATP concentrations of 0-8 mM and 2 mM 2,3-DPG (B). .
• : Long-term stability measurement of the recombinant cDPGS. The enzyme was stored at -80°C in a buffer consisting of MES/KOH pH 6.5, 25% (v/v) glycerol, 400 mM KCl, 5 mM MgCl ₂ and 10 mM DTT. Residual activity was determined at the times indicated using the continuous assay as described in Examples (Materials and Methods). 100% of the relative activity corresponds to a specific activity of 13.5 U mg ^-1 .
• : Kinetics of cDPGS-mediated conversion of 2,3-DPG to cDPG. The ATP and 2,3-DPG dependent production of cDPG was determined in a laboratory scale conversion assay. The time-dependent consumption of 2,3-DPG (inverted triangles) was analyzed using the 2,3-DPG assay kit from Roche (Mannheim, Germany) according to the manufacturer's instructions but modified for 96-well plates. The corresponding amount of ADP produced from ATP (triangles) was calculated from the respective reactions coupled to the oxidation of NADH using rabbit muscle pyruvate kinase (PK) and lactate dehydrogenase (L-LDH) as auxiliary enzymes, as in the examples ( Material and methods) described, calculated.

Detailed description

The present invention is based on results of the successful expression and isolation of cyclic 2,3-diphosphoglycerate synthetase (cDPGS) in a mesophilic host organism and use for an efficient one-step in vitro synthesis of cyclic 2,3-diphosphoglycerate.

The invention relates to methods for the recombinant production of a polypeptide from a thermophilic or hyperthermophilic organism in mesophilic host organisms.

The term "thermophilic" as used herein refers to organisms whose growth optimum is in an (ambient) temperature range of >60°C. Similarly, the term "hyperthermophilic" as used herein refers to organisms whose growth optimum is in a temperature range of >80°C. In contrast, “mesophilic” organisms are those that thrive optimally in a temperature range of <60°C, particularly in temperature ranges of 10 to <60°C, or 10 to 50°C, or 10 to 42°C, typically in the range of 20 to 40°C.

In the methods of the invention, in a first step a) a nucleic acid molecule encoding the polypeptide of interest is introduced into the mesophilic host organism. The nucleic acid molecule can be the native sequence, but in various embodiments this is suitably modified, for example codon-optimized, for expression in the host organism. Corresponding methods and options for codon optimization in order to achieve expression in a specific host organism that is as problem-free and strong as possible are known to the person skilled in the art and corresponding commercial offers are routinely available. The coding nucleic acid sequence can be used as such or as part of a larger molecule, for example as part of an expression vector which can also be present as a plasmid. In such molecules, the actual coding sequence is often combined with other functional sequences, such as selection markers and regulatory elements, the duplication and expression in the Regulate and control the host organism and, if necessary, also the stable incorporation into the genome of the host organism. Also possible is functional linking to a further sequence which codes for a peptide or polypeptide tag which can simplify the purification and isolation of the polypeptide. In various embodiments, however, it can be advantageous not to use such tags. On the one hand, in some cases these are not necessary for a successful purification, on the other hand, this avoids the need to split them off again at a later point in time. Depending on the host organism, the nucleic acid molecule can be introduced using methods known per se, for example transformation by means of heat shock, which can be carried out in E. coli at a temperature of 41-43° C. and in the presence of calcium chloride. Other transformation and transfection methods are known per se to those skilled in the art and can also be used.

In a further step of the method, the mesophilic host organism is then cultivated under conditions which allow the expression of the polypeptide of interest. These conditions are usually temperature controlled and often require the use of a suitable growth and expression medium. Suitable media are known in the art and include, for example, E. coli LB medium for the host organism. Since the host organism is a mesophilic organism, the cultivation conditions are typically limited to a temperature range of up to 42°C, with the optimum being in the range from 10 to 42°C. While higher temperatures often result in faster growth and stronger expression, and the cultivation times can be shortened as a result, it can be advantageous for some polypeptides to choose lower temperatures in order to obtain stable and soluble polypeptides. In various embodiments of the expression of cyclic 2,3-diphosphoglycerate synthetase (cDPGS) in E. coli, cultivation and incubation temperatures of 16 to 30° C. were used, for example 30° C. or 16° C., optimal results being obtained at a temperature of 16°C were obtained.

All organisms used in the prior art for protein expression can be considered as mesophilic host organisms, such as in particular E. coli or various Bacillus species, but also eukaryotic expression systems, such as those from yeasts or fungi. The mesophilic host organism is in particular a prokaryote, in particular a bacterium. In various embodiments, the host organism is a special expression strain that was developed specifically for the expression of heterologous polypeptides or is suitable for this.

In various embodiments, the addition of an inducer may also be necessary to start expression, with this depending on the use of special regulatory sequences on the nucleic acid used. A known inducer for expression in E. coli is, for example, IPTG (isopropyl-beta-D-thiogalactopyranoside), which is used together with the lac operon to induce it.

After expression of the polypeptide has taken place, the next step c) is the isolation of the polypeptide from the mesophilic host organism. For this purpose, the host organism is typically lysed in a first step. This lysis can be carried out using known techniques, for example ultrasound, homogenization in a homogenizer, chemical lysis or in a French press apparatus. The lysate obtained is typically centrifuged to separate the solid cell components from the soluble components in the supernatant. Since the expressed polypeptide originates from a thermophilic or hyperthermophilic organism, it is usually significantly more heat-stable than the proteins of the host organism. This fact can be exploited during isolation in such a way that either the lysate itself or the supernatant obtained after centrifugation of the lysate is heated. This leads to the precipitation of the non-heat-stable proteins from the supernatant. Suitable temperatures are in the range from 50 to 95°C, for example 60 to 90°C, the specific temperature used depending on the type of polypeptide being expressed. For example, lower temperatures tend to be used for polypeptides from thermophilic organisms than for those from hyperthermophilic organisms. In general, however, temperatures of around 60°C are already sufficient to precipitate all undesired proteins of the host organism dissolved in the supernatant. In various designs, however, temperatures of 65° C. and more, for example 70° C., 75° C. or 80° C., can also be used. Usual incubation times at these elevated temperatures range from 5 to 60 minutes, for example 10 to 40 minutes, or 20 to 30 minutes. In order to separate the still dissolved protein of interest from the foreign proteins thus precipitated, a (further) centrifugation step is normally carried out. The supernatant obtained in this way often already contains the desired protein in a comparatively pure form.

However, it is possible and, in various embodiments, also advantageous to add further purification steps. come at it in particular chromatographic methods are used. If the polypeptide was expressed with an affinity tag, a special affinity chromatography can be used, for example. Known tags include, without limitation, 6xHis tags, GST tags, and MBP tags. Chromatography materials suitable for such tags, which enable purification, are commercially available. Other chromatographic methods include, in particular, size exclusion chromatography, but ion exchange chromatography may also be suitable.

Expression and purification can be checked, for example, using SDS-PAGE.

The expressed polypeptide from the thermophilic or hyperthermophilic organism is - in various embodiments - an enzyme. Suitable enzymes include, but are not limited to, EC class 2, 3, 4 and 6 enzymes such as glycosyltransferases (EC class 2), hydrolases (EC class 3), lyases (EC class 4) and ligases (EC class 6) . In various embodiments, the enzymes are selected from those of the classes esterases, lipases, glycoside hydrolases, dehydratases, aldolases, decarboxylases and synthetases. Those which catalyze at least one step in the production of an extremolyte are particularly preferred. This synthetic step is usually a key step in the synthesis, allowing the desired substance to be produced from a readily available substrate. If several steps are required for this, the corresponding enzymes can be produced and isolated using the methods described herein and then used for the respective synthesis step. The product from a first step can then be used for a further enzymatic synthesis step.

In various embodiments, the polypeptide is an enzyme selected from trehalose glycosyl-transferring synthase (TreT) and cyclic 2,3-diphosphoglycerate synthetase (cDPGS), in particular the cDPGS from Methanothermus fervidus and functional variants or fragments thereof. In various embodiments, the cDPGS comprises or has the amino acid sequence given in SEQ ID NO:1.

The term "fragments", as used in this context, refers to fragments of the full-length sequence which, however, have substantially (i.e. at least 80% relative to the full-length sequence) retained their functionality. Such fragments are often N- and/or C-terminally shortened sequences which differ from the full-length sequence in that the ends are shortened by 1 to 50 amino acids, e.g. 1 to 20 or 1 to 10 amino acids. Since the ends are often unstructured and flexible, such shortening often does not affect the functionality of the enzyme.

The term "functional variants" as used herein refers to variants that retain the functionality of the parent enzyme, typically at least 80% of it, but differ from the parent sequence by one or more sequence differences. The sequence identity of such variants may be in the region of 80% based on the total length of the variant and/or parent enzyme, and may be at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, or 99%.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm, which is established and commonly used in the prior art (cf., for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool ." J. Mol. Biol. 215:403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): " Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, p.3389-3402) and is done in principle by assigning similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another become. A tabular assignment of the relevant positions is referred to as an alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created with computer programs. For example, the Clustal series (cf., for example, Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500), T-Coffee (cf., for example, Notredame et al (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217) or programs based on these programs or algorithms. Sequence comparisons (alignments) are also possible using the Vector NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW. Unless otherwise indicated, sequence identity given herein is determined using the BLAST algorithm.

Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually given as a percentage of identity, ie the proportion of identical nucleotides or amino acid residues in the same positions or in positions that correspond to one another in an alignment. In the case of amino acid sequences, the broader concept of “homology” includes conserved amino acid exchanges, i.e. amino acids with similar chemical activity, since these usually have similar chemical activities within the protein. Therefore, the similarity of the compared sequences can also be expressed as percent homology or percent similarity. Identity and/or homology statements can be made about entire polypeptides or genes or only about individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences. Unless otherwise stated, information on identity or homology in the present application relates to the total length of the nucleic acid or amino acid sequence indicated in each case.

In the methods of the invention, the thermophilic or hyperthermophilic organism from which the polypeptide to be expressed is derived may be a prokaryote, for example selected from bacteria and archaea.

When the organism of origin is a bacterium, this can be selected, for example, from bacteria of the orders Thermales, Thermotogales and Aquiificales. Further examples are bacteria of the genera Thermus, Thermotoga and Aquifex and the corresponding species Thermus spp., Thermotoga spp. and Aquifex spp.

Alternatively, the polypeptide can also be derived from Archaea of the lines Crenarchaeota and Euryarchaeota. In the case of the Archaea of the lines Crenarchaeota and Euryarchaeota, organisms of the classes or orders Thermoprotei, Desulfurococcales, Sulfolobales, Methanopyri, Methanococci, Methanobacteria, Thermococci, Thermoplasmata and Archaeoglobi are particularly suitable. Thermoproteales, Pyrodictium and Sulfolobus come into consideration here, as well as Thermococcales, Thermoplasmatales, Archaeoglobales, and thermophilic methanogens of Methanopyrales, Methanococcales, Methanobacteriales, in particular of the genera Thermoproteus, Pyrodictium, Sulfolobus, Methanopyrus, Methanocaldococcus, Methanococcus, Methanothermobacter, Methanobacterium, Methanothermus, Thermococcus, Pyrococcus , Thermoplasma, Picrophilus, Ferroplasma, Archaeoglobus and their respective associated species (spp.). In one embodiment, the organism is Methanothermus fervidus, Pyrococcus woeseii, or Pyrococcus furiosus.

In various embodiments of the invention, the expressed polypeptide is Methanothermus fervidus cyclic 2,3-diphosphoglycerate synthetase (cDPGS). In such embodiments, the host organism is in particular E. coli. In various embodiments, the nucleic acid coding for cDPGS (SEQ ID NO:2) is codon-optimized for use in E. coli and can therefore have, for example, the nucleotide sequence according to SEQ ID NO:3. It was found that the expression of cDPGS in E. coli has an optimum in a temperature range around about 16°C. Cultivation and expression therefore preferably take place at this temperature. It has also been found that isolating the enzyme in active form requires elevated salt concentrations. The methods described herein, in particular the isolation steps, therefore preferably take place in the presence of elevated salt concentrations of 250 mM and more, for example 300 mM and more or 400 mM and more. Potassium chloride is particularly suitable as the salt. The isolation is therefore preferably carried out in the presence of 400 to 500 mM KCl. The optimal pH for purification is in the range from 5.5 to <7, for example in the range from 6 to 6.8.

It was also found for the cDPGS from Methanothermus fervidus that it could not be expressed stably with an affinity tag, in particular a 6x His tag. The methods described herein are therefore preferably carried out for this enzyme without the use of an affinity tag which is fused to the enzyme.

In a further aspect, the invention relates to a method for the enzymatic production of extremolytes, the method comprising the use of recombinantly produced enzymes from hyperthermophilic organisms which can be obtained according to one of the methods of the invention. Such methods may in particular be in vitro methods in which the isolated enzyme is contacted with one or more suitable substrates under conditions which permit enzymatic activity. The enzymes used in these methods can be any of the enzymes described herein in the context of the expression methods.

In various embodiments of such methods, the extremolyte is cyclic 2,3-diphosphoglycerate (cDPG) and the polypeptide/enzyme is cyclic 2,3-diphosphoglycerate synthetase (cDPGS), in particular the cDPGS from Methanothermus fervidus as defined above. cDPG is synthesized starting from 2,3-diphosphogly cerate as substrate and in the presence of ATP and magnesium ions in a temperature range adapted for the enzyme, for example at temperatures in the range from 50 to 70°C, such as 55 or 60°C. Alternatively, a two-step synthesis can be carried out starting from 2-phosphoglycerate (2PG), which also requires the use of 2-phosphoglycerate kinase (2PGK) ( ). However, since the substrate 2,3-DPG is more easily and cheaply available than 2PG, a one-step synthesis using only cDPGS is preferred.

In yet another aspect, the invention relates to the use of the polypeptides produced by one of the methods of the invention for the production of extremolytes, in particular for the in vitro production of extremolytes. In such uses, the polypeptide may be an enzyme as defined in the context of the expression methods of the invention, for example cDPGS from Methanothermus fervidus. In these embodiments, the extremolyte is then cDPG.

examples

material and methods

Cloning, heterologous expression and purification of the recombinant proteins

cloning

The M. fervidus 2,3-diphosphoglycerate cyclic synthetase (cdpgs, 1383 bp) gene (Mfer_0077, CAA70986) was amplified by PCR from M. fervidus chromosomal DNA (the primer pairs for cloning are given in listed). The PCR products were cleaned using the Wizard® SV Gel and PCR Clean-Up System (Promega, Fitchburg, USA) according to the manufacturer's instructions. Cut DNA fragments were cloned into the expression vector pET24a (Kan ^R ) and pET15b (Amp ^R ) (Novagen, Merck, Darmstadt, Germany) with a C- or N-terminal His tag and in E. coli DH5α (Life Technologies, Carlsbad , USA). Furthermore, the cdpgs gene was codon-optimized for expression in E. coli (Eurofins, Ebersberg, Germany) and subcloned into pET15b (Amp ^R ) without an affinity tag (the primer pairs and plasmids are in listed). All sequences and plasmids were verified by sequencing (LGC Genomics, Berlin, Germany).

Heterologous Expression

Competent cells of E. coli BL21(DE3)-CodonPlus-pRIL (Cam ^R ) (Merck, Darmstadt, Germany) were transformed with the constructed plasmids and placed in 200 mL - 400 mL LB medium (lysogeny broth medium) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Expression was induced by the addition of 1mM isopropyl-β-d-thiogalactopyranoside (IPTG) at an OD ₆₀₀ between 0.6 - 0.8. Cultures were incubated overnight at 30°C and at 16°C for optimized results. The cells were harvested (6,000×g, 20 min, 4° C.) and per 1 g wet cell mass in 5 mL buffer (50 mM MES (2-(N-morpholino)ethanesulfonic acid), 5 mM DTT, 2 mM MgCl ₂ , 300- 500mM KCl, pH 6.5). For cell lysis, the cell suspensions were passed three times through a French press cell at 20,000 psi (about 1379 bar). The cell lysate was centrifuged (25,000xg, 45 min, 4°C) to remove all cell debris. The supernatant containing the recombinant, thermophilic cDPGS was subjected to a heat precipitation step at 75°C for 30 min (Eppendorf Thermomixer at 700 rpm), followed by centrifugation (25,000xg, 45 min, 4°C) to remove denatured, mesophilic remove host proteins.

cleaning

The protein fractions obtained after the heat treatment were further purified. For affinity chromatography, the supernatant was applied to a Protino® Ni-TED column (Tris(carboxymethyl) ethylenediamine 1000 or 2000) (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and, according to the manufacturer's instructions, using 50 mM Tris/HCl or 50mM potassium phosphate pH 8 buffer (50mM Tris or 50mM K ₂ HPO ₄ /KH ₂ PO ₄ , 5mM DTT, 300mM KCl or 300mM NaCl, 250mM imidazole). Protein dialysis was performed using Spectra/Por® dialysis membrane standard RC tubing (Repligen, Waltham, USA) with a molecular weight cut-off of 30 kDa. The optimized protein, which does not contain an affinity tag, was equilibrated in 50 mM MES/KOH, 300 mM KCl, pH 6.5 by size exclusion chromatography (SuperdexTM 200, HiLoadTM 26/60 column, 320 ml volume, GE Healthcare Life Science, Freiburg, Germany). (flow rate 2 mL/min)) ( ). The expression, purification and molecular masses of the subunits were monitored by denaturing SDS-polyacrylamide gel electrophoresis. Protein concentration was determined by a modified Bradford method using bovine serum albumin as a standard (Bio-Rad, Feldkirchen, Germany).

Determination of cDPGS activity in a continuous assay and determination of 2,3-DPG consumption and cDPG formation in a discontinuous assay system

enzyme properties

The enzyme activity was determined by coupling the ADP formation from ATP to the oxidation of NADH by pyruvate kinase (PK) and L-lactate dehydrogenase (LDH) (rabbit muscle, Merck, Darmstadt, Germany). The experimental batches (0.5 mL) contained 50 mM MES/KOH pH 6.5 with 2.5 mM MgCl ₂ , 400 mM KCl, 2.5 mM ATP, 0.5 mM NADH, 3.7 µg purified cDPGS, 8 U PK, 4 U LDH and 2 mM phosphoenolpyruvate. After 1 min pre-incubation at 55°C, the reactions were started by adding 2,3-diphosphoglycerate (2,3-DPG) and the oxidation of NADH was measured in a Specord 210 UV/VIS spectrometer (Analytic Jena AG, Jena, Germany) tracked. All measurements were performed in triplicate.

Influence of salt and pH

The stimulation of enzyme activity was analyzed in the presence of KCl. For this purpose, the enzyme activity was analyzed, as described above, with different salt concentrations up to 500 mM and 16.8 μg of partially purified cDPGS after the heat treatment. In addition, the pH optimum was maintained in a range of pH 5.5 - 8.0 using 50mM MES/KOH pH 5.5 and 6.5, 50mM TES/KOH pH 6.8, 50mM potassium phosphate buffer pH 6 .8 and HEPES/KOH pH 7.0 and 8.0 with the addition of 400 mM KCl.

Consumption of 2,3 DPG and formation of cDPG

cDPG synthesis was performed in a batch assay (1mL) in 50mM MES/KOH pH 6.5, 2.5mM MgCl ₂ , 2.5mM ATP, 1mM 2.3DPG, 400mM KCl at 55° C measured using 7.4 µg purified cDPGS. 100 µL samples were withdrawn into pre-cooled reaction tubes at regular intervals and stored directly at -80°C to stop the reaction. The amount of 2,3-DPG consumed was determined in 10 µL of these samples using a 2,3-DPG test kit from Roche (MERCK, Darmstadt, Germany). The assay was performed according to the manufacturer's instructions, but modified for 96-well plates in a total volume of 200 µl using an NADH standard curve obtained at 340 nm using the Tecan Microplate Reader (Tecan Group Ltd., Lifesciences, Männedorf, Switzerland). . Similarly, the amount of ADP resulting from ATP was determined in 50 µL of these samples from the same time interval using the PK and LDH assay conditions described above.

Example 1: Expression and purification of M. fervidus cDPGS

Heterologous expression of M. fervidus cyclic 2,3-diphosphoglycerate synthetase (cdpgs) in E. coli results in only low levels of the soluble protein ( ). Because the codon adaptation index for cDPGS expression in E. coli was low (CAI=0.54), the gene was codon-optimized for expression in E. coli (Eurofins, Ebersberg, Germany). This resulted in higher amounts of soluble protein ( ). However, the His-tagged cDPGS obtained from these expressions was rather unstable in solution. Therefore, the codon-optimized gene was expressed without an affinity tag in E. coli BL21(DE3)-CodonPlus-pRIL(Cam ^R ) (MERCK, Darmstadt, Germany) and additionally the expression temperature was lowered to 16°C. After overnight expression, the enzyme was purified by heat precipitation (75°C, 30 min) followed by size exclusion chromatography. 3.5 mg pure protein were obtained from 400 mL expression culture, which corresponds to 1.7 g wet cell weight ( ). The molecular weight of cDPGS was 52 kDa under denaturing conditions determined by SDS-PAGE ( ), and 119 kDa under native conditions determined by size exclusion chromatography, indicating a homodimeric structure of the protein.

Example 2: Characterization of M. fervidus cDPGS

cDPGS catalyzes the ATP-dependent formation of an intramolecular phosphoanhydride bond in 2,3-DPG, resulting in cyclic 2,3-diphosphoglycerate (cDPG). Activity was determined by monitoring ADP formation using pyruvate kinase (PK) and L-lactate dehydrogenase (LDH) (rabbit muscle, Merck, Darmstadt, Germany). However, it could be shown that cDPGS activity was strongly salt-dependent: a 5.6-fold activation was observed in the presence of 500 mM KCl ( . The pH optimum of cDPGS was pH 6.5 and the optimal buffer for characterization was 50mM MES/KOH with 400mM KCl, 2.5mM MgCl ₂ and 10mM DTT ( . The enzyme activity/(co)substrate dependence of the pure cDPGS followed classical Michaelis-Menten kinetics with a V _max of 21.7 U mg ^-1 and K _m values of 1.4 mM and 1.1 mM for 2,3- DPG and ATP ( & B, sat ).

The purification steps were performed in the presence of 400 mM KCl, 2.5 mM MgCl ₂ and 10 mM DTT as reducing agents. The stability of cDPGS could be significantly improved under these conditions. For storage at -80°C, 25% (v/v) glycerol was added to the buffer to preserve enzyme activity. After 1 week of location At -80°C, the enzyme remained 100% active and even after 1.5 months almost full activity (95%) was retained ( ).

Example 3: cDPG production

The in vitro synthesis of cDPG from 2,3-DPG was analyzed at 55°C on a small scale. For this, 1 mM 2.3 DPG (0.76 mg in 1 mL) with 2.5 mM ATP in 50 mM MES/KOH pH 6.5 containing 400 mM KCl, 2.5 mM MgCl ₂ , 10 mM DTT and 7 .4 µg of the purified cDPGS (corresponds to 0.11 U) incubated. The consumption of 2,3-DPG was monitored in a discontinuous assay using the modified assay procedure of the 2,3-DPG test kit from Roche (Merck, Darmstadt, Germany) as described above. After 10 min of incubation, 56% of 2,3-DPG was converted by the cDPGS, after 30 min 92%, and after 60 min the 2,3-DPG was completely consumed ( ). As a control, the non-enzymatic hydrolysis of ATP was determined at 55°C under the same conditions but without the recombinant cDPGS. It turned out that only 4.5% of the ATP was lost after 60 min due to non-enzymatic hydrolysis. Taken together, these results indicate a complete conversion of 2,3-DPG to cDPG by the recombinant M. fervidus cDPGS.

While particular embodiments of the present invention have been shown and described, it would be apparent to those skilled in the art that various other changes and modifications can be made without departing from the scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications as fall within the scope of the invention.

A sequence listing follows as an electronic document. This can be accessed both in DEPATISnet and in the DPMAregister.

Claims (14)

A method for the recombinant production of a polypeptide from thermophilic or hyperthermophilic organisms in mesophilic host organisms comprising a) introducing a nucleic acid molecule which encodes the polypeptide from a thermophilic or hyperthermophilic organism into the mesophilic host organism; b) culturing the mesophilic host organism under conditions allowing expression of the polypeptide; and c) isolation of the polypeptide from the mesophilic host organism. procedure after claim 1 , characterized in that the nucleic acid molecule coding for the polypeptide from a thermophilic or hyperthermophilic organism is codon-optimized for expression in the mesophilic host organism. procedure after claim 1 or 2 , characterized in that the cultivation of the host organism takes place under mesophilic conditions, in particular in a temperature range between 10 and 45°C. Procedure according to one of Claims 1 until 3 , characterized in that the isolation of the polypeptide from the mesophilic host organism comprises: a) lysis of the host organism; b) heating the lysate from step a) or a supernatant obtained from the lysate to a temperature of 50 to 95°C, preferably 60 to 90°C, to precipitate the host proteins and polypeptides; and c) isolating the soluble supernatant from the precipitate obtained in step b). procedure after claim 4 , characterized in that the method further comprises isolating the polypeptide from the solvate obtained in step c), in particular by means of size exclusion chromatography. The procedure according to one of the Claims 1 until 5 , characterized in that the polypeptide comprises no affinity tag, preferably no non-native peptide sequence. Procedure according to one of Claims 1 until 6 , characterized in that the polypeptide is an enzyme, in particular an enzyme which catalyzes at least one step in the production of an extremolyte. procedure after claim 7 , characterized in that the enzyme is selected from glycosyltransferases (EC class 2), hydrolases (EC class 3), lyases (EC class 4) and ligases (EC class 6), in particular esterases, lipases, glycoside hydrolases, dehydratases, aldolases, decarboxylases and synthetases. The procedure according to one of the Claims 1 until 8th , characterized in that the polypeptide is selected from the trehalose glycosyl-transferring synthase (TreT) and the cyclic 2,3-diphosphoglycerate synthetase (cDPGS), in particular the cDPGS from Methanothermus fervidus and functional variants or fragments thereof. Procedure according to one of Claims 1 until 9 , characterized in that the thermophilic or hyperthermophilic organism is a prokaryote selected from bacteria and archaea, preferably from bacteria of the orders Thermales, Thermotogales and Aquiificales or Archaea of the lines Crenarchaeota and Euryarchaeota. procedure after claim 10 , characterized in that the organism is a representative of the Archaea of the lineages Crenarchaeota and Euryarchaeota and is selected from one of the classes Thermoprotei, Methanopyri, Methanococci, Methanobacteria, Thermococci, Thermoplasmata and Archaeoglobi. Method for the enzymatic production of extremolytes, the method comprising the use of recombinantly produced enzymes from thermophilic or hyperthermophilic organisms according to one of the methods according to claims 1 - 11 are available. The procedure according to claim 12 wherein the extremolyte is cyclic 2,3-diphosphoglycerate (cDPG) and the polypeptide is cyclic 2,3-diphosphoglycerate synthetase (cDPGS). Use of the polypeptides produced according to any one of Claims 1 until 11 for the production of extremolytes, in particular for the in vitro production of extremolytes.