GB2501882A - Method for producing cyanophycin - Google Patents

Abstract

A method for producing cyanophycin in a recombinant microorganism comprises providing a nitrogen source for the microorganism, and conversion of the nitrogen source into cyanophycin, wherein the microorganism expresses a recombinant P(II) signal transduction protein variant with increased binding to N-acetyl-L-glutamate kinase (NAGK). The superactive NAGK binder may be a variant P(II) signal transduction protein from Synechococcus elongatus PCC 7942 with an I86N or I86T mutation. The recombinant microorganism may further comprise a heterologous NAGK gene (if it does not naturally express NAGK) and optionally other genes involved in cyanophycin biosynthesis such as cyanophycin synthetase (CphA). Preferably, said recombinant microorganism is selected from a diazotrophic microorganism able to fix atmospheric nitrogen, e.g. heterocyst-forming cyanobacteria such as Anabaena. The invention further relates to recombinant strains comprising P(II) variants and the use of said recombinant bacterial strains for the production of cyanophycin, preferably using atmospheric nitrogen as the nitrogen source.

Description

Method for producing cyanophycin The present invention relates to a method for producing cyanophycin in a recombinant micro-organism, comprising a) providing a recombinant microorganism, b) providing a nitrogen source for said recombinant microorganism, and c) conversion of said nitrogen source into cyanophycin by said recombinant microorganism, wherein said recombinant microorganism expresses a recombinant P(II) signal transduction protein variant that is a superactive N-Acctyl-L-Glutamate (NAGK) protein binder. Preferably, said recombinant microorganism is selected from a diazotrophic microorganism able to fix atmospheric nitrogen. The present invcntion further relates to rccombinant strains comprising P(II) variants and the use of said recombinant bacterial strains for the production of cyanophycin, preferably using atmospheric nitrogen as the nitrogen source or one of the nitrogen sources.

Background of the invention

Cyanophycin, or multi-L-arginyl-poly (L-aspartic acid), is an amino acid polymer composed of an aspartic acid backbone and arginine side groups. Cyanophycin was first discovered in 1887 and can be found in most cyanobacteria and a few heterotrophic bacteria such as Ac/ne-tobacter sp.

Cyanophycin is largely insoluble under physiological conditions and is accumulated in the form of granules in the cytoplasm during phosphate or sulfur starvation, generally in the early and mid-stationary phase. It is used as a nitrogen-and possibly carbon-storage compound and also serves as a dynamic buffer for fixed nitrogen in cyanobacterial heterocysts. Nitrogen and carbon are mobilized from cyanophycin by intracellular cyanophycinase in the form of aspar-tate-arginine dipeptides.

Cyanophycin is synthesized from arginine and aspartate in an ATP-dependent reaction cata-lyzed by a single enzyme, cyanophycin synthetase. Several publications disclose the isolation of the cyanophycin synthetase genes, e.g. from Synechocystis PCC 6803 or Anabaena van- abilis ATCC 29413 (DE-A 19813692) allowing for the production of cyanophycin in recom-binant bacteria.

Cyanophycin is of potential interest to biotechnology as a source of polyaspartic acid. Het- erologous expression of cyanophycin synthetase allows production of cyanophycin in a num-ber of biotechnological ly relevant bacteria such as Eschertc/iia colt and Corvnehacterium glutwn icutn.

Cyanophycin can also be produced in transgenie plants, such as potatoes. The eyanophycin and its components polyasparate and arginine so produced can be used as degradable plastics, for the softening of water, and food additives.

The P(ll) proteins constitute one of the largest and most widely distributed family of signal transduction proteins present in archaea, bacteria, and plants. P(II) signal transduction pro-teins are highly conserved and control key processes of nitrogen metabolism in response to central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and car-bon and nitrogen abundance. These effectors bind to P11 in an interdependent manner, thereby transmitting metabolic information into structural states of this sensor protein.

In cyanobacteria and plants, the controlling enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase (NAGK), is a major PUT) target. Depending on its effector molecule binding status, P01) (from cyanobacteria and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic omithine pathway, N-acetyl-1-glutamate kinase (NAGK), to control arginine biosynthesis. Moreover, PUT) affects gene expression in cyano-bacteria through binding to the transcriptional coactivator of NtcA, PipX.

Fokina et al. (Fokina 0, Chellamuthu YR, Zeth K, Forchhammer K. A novel signal transduc- tion protein P01) variant from Synechococcus elongatus PCC 7942 indicates a two-step proc-ess for NAGK-P(II) complex formation. J Mol Biol. 2010 Jun 1 1;399(3):410-21. Epub 2010 Apr 24) describe P01) variants with altered binding characteristics and found POT) variants 186N and 186T to be able to bind to an NAGK variant R233A) that was previously shown to be unable to bind wild-type P01) protein. Analysis of interactions between these P01) variants and wild-type NACK as well as with the NAGK R233A variant suggested that the PUT) 186N variant was a superactive NAGK binder. Both P01)186 variants displayed a specific defect in 2-OG binding, implying a role of residue 186 in 2-OG binding.

Furthermore, Fokina et a!. (Fokina 0, Chellamuthu YR, Forchhammer K, Zeth K. Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus Pit signal transduction protein.

Proc Nat! Acad Sci U S A. 2010 Nov 16;107(46):19760-5. Epub 2010 Nov 1.) describe struc-tures of the Syneehocoecus elongatus P01) protein in complex with ATP, Mg(2+), and 2-0G.

which clari' how 2-OG affects P(TI)-NAGK interaction.

US2004002053 describes plant nitrogen regulatory P11 genes, nucleotide sequences, expres-sion constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P11 gene from said constructs.

US2009036576 describes fermentation processes for the production of cyanophycin in a mi-croorganism whereby a plant-derived nitrogen source is converted by the microorganism into cyanophycin. The description further relates to processes for the conversion of cyanophyein into a variety of compounds, preferably nitrogen-containing compounds. US2009036576 thus provides for methods for valorising N-containing waste streams of plant materials.

Hai et al. (in Hai T, Frey K1VI, Steinbilehel A. Engineered eyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and eyanophycin accumulation to Escherichia coli. Appl Environ Mierobiol. 2006 Dec;72(12):7652-60. Epub 2006 Sep 29) de-scribe the cyanophycin (CGP) synthetase gene (cphANEl) of the transposon-induced argL mutant NEI of the eyanobaeterium Nostoe ellipsosporum, which exhibits a CGP-leaky phe-notype during diazotrophical growth, and its cloning and expression in Eseheriehia coli strain TOP 10. Recombinant cells of E. coli accumulated CGP up to 17 and 8.5% (wtlwt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-beta-D-thiogalactopyranoside, respectively. A truncated CphA, lacking 31 CphANE1del96) amino acids of the C-terminal region, was derived from cphANEl by deleting 96 from its 3' region through the introduction of stop eodons. In comparison to the wild-type gene, the cells accumulated about twofold more CGP (up to 34.5% wtIwt] of CDM) than cells expressing the wild-type gene.

Finally, Elbahloul and SteinbUchel (Engineering the genotype of Acinetobaeter sp. strain ADP1 to enhance biosynthesis of eyanophycin. App! Environ Microbiol. 2006 Feb;72(2): 1410-9) describe a study of the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophyein (CGP) in Aeinetobacter sp. strain ADPI, where genes encoding the putative arginine regulatory protein (argR) and the arginine succinyl transferase (astA) were inactivated, and the effects of these mutations on CGP syn-thesis were analyzed. The inactivation of these genes resulted in a 3.5-or 7-fold increase in COP content, respectively, when the cells were grown on glutamate. Overexpression of ArgF (ornithine carbamoyl transferase), CarA-CarB (small and large subunits of carbamoylphos-phate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohy- drate metabolism, showed a significant increase in CGP contents when grown on mineral me-dium supplemented with glutamate, aspartate, or arginine. The Acinetobaeter sp. Delta astA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon.

In summary, despite the above approaches, still an economically feasible production of cyanophycin by fermentation is desired, as using the constituting amino acids arginine and aspartate is far too expensive when these amino acids are obtained by sugar-based fermenta-tion or enzymatic catalysis, respectively. On the other hand, the fermentation yield on sugar and ammonia is too low. There is thus still a need in the art for an economically feasible route to the production of cyanophycin for high-value specialty applications of eyanophycin, such as in medical or surgical devices, (food) packaging materials and coatings. It is therefore an object of the present invention, to provide such a method. Other objects and advantages will become apparent when studying the appended claims as well as the detailed description of the invention as follows.

According to a first aspect of the present invention, the above object is solved by a method for producing cyanophycin in a recombinant microorganism, comprising a) providing a recombi-nant microorganism, b) providing a nitrogen source for said recombinant microorganism, and c) conversion of said nitrogen source into cyanophycin by said recombinant microorganism, wherein said recombinant microorganism expresses a recombinant P(II) signal transduction protein variant that is a superactive N-Acetyl-L-Glutamate (NAGK) protein binder.

Preferably, said recombinant microorganism is selected fix,m a diazotrophic microorganism able to fix atmospheric nitrogen, such as, for example, heterocyst-forming cyanobacteria, such as Anabaena; non-heterocystous cyanobacteria such as Cyanothecae and heterotrophic nitrogen-fixing bacteria, such as Rhizobia; Frankia; and Azotobacteraceae.

A second aspect of the present invention then relates to a recombinant strain of a microorgan-ism that naturally contains NAGK protein, preferably selected from Cyanobacteria, such as Aphanocapsa, Synechococcus, Synechocyctis, Anabaena, and Spirulina, and preferably Ana- baena, comprising a P(ll) signal lransduction protein variant that is a superactive NAGK pro-tein binder, which is preferably selected from the protein P(lI) variant from Synechococcus elongatus PCC 7942 186N, and 186T.

A preferred third aspect of the present invention then relates to the use of a recombinant bac-terial strain according to the present invention for the production of cyanophycin, preferably using atmospheric nitrogen as the nitrogen source or one of the nitrogen sources.

As mentioned above, the present invention is directed at a new and improved method for pro- ducing cyanophycin in a recombinant microorganism. Said recombinant microorganism ex- presses a recombinant P(ll) signal transduction protein variant that is a superactive N-Acetyl-L-Glutamate (NAGK) protein binder. In its preferred embodiment, the present invention thus is based on the surprising finding that the production of cyanophycin in a microorganism can be increased by modHing the P(II) signal transduction protein in a way to produce a protein variant that exhibits an increased binding towards the NAGK protein. This binding step seems to be the rate-limiting step in thc cyanophycin synthesis, and therefore an increased binding leads to an increase of cyanophycin production in said microorganism.

A P(ll) signal transduction protein "variant" is a P(ll) signal transduction protein that is modi- fied in a way to exhibit an increased (e.g. stronger) binding towards the NAUK protein. Pre-ferred are variants that have been genetically altered, and, for example, contain mutations that lead to amino acid deletions, insertions, and/or replacements, such as, for example, mutations that lead to a defect in 2-00 binding of the P(ll) signal transduction protein variant, which is preferably selected from the protein P(ll) variant from Synechococcus elongatus PCC 7942 186N, and 186T.

This and other exemplary P(II) signal transduction proteins that can be used in the context of the present invention are listed in the following table: Organism Database Ace. No. Synechococcus sp. (P11-variant) AAA273 12.1 Nodularia spumigena EAW44623.1 Anabaena variabilisATCC 29413 ABA19763.1 Nostoc sp. PCC 7120 NP 486359.1 Fischerellasp. JSC-l I EHCO89OI.l Tolypothrix sp. PCC 7601 CAA65992.1 Nostoc azollae' 0708 ADl62691.l Nostoc punctiformePCC 73102 ACC82833.1 Raphidiopsis brookii D9 EFA74248.1 Cylindrospermopsis raciborski i CS-SOS EFA6978 1.1 Oscillatoria sp. PCC 6506 CBNSS7I6.1 Lyngbya sp. PCC 8106 EAW36368.l Arthrospira sp. PCC 8005 CCE18580.1 Microcoleus chthonoplastes PCC 7420 EDX78595.1 Trichodesmium erythraeum IMS1OI ABG52016.1 Synechococcus sp. PCC 7335 EDX83986.1 Cyanothece sp. PCC 7425 ACL4508 1.1 Acaryochloris marina MBICI 1017 ABW28154.1 A "superactive" N-Acetyl-L-Glutamate (NAGK) protein binder is any P(II) signal transduc-tion protein variant that exhibits an increased (e.g. stronger) binding to the NAGIC protein, when compared to an unmodified P01)-protein, preferably of the same origin. Superactive according to thc prescnt invcntion can also include an increased amount of P(II) signal trans-duction protein, based on an increased expression such as, for example, overexpression of said P01) signal transduction protein in a recombinant cell. Then, said cell preferably contains more than 10%, preferably more than 25% and most preferred more than 50% more of said P01) signal transduction protein, compared to a non-increased expression oroverexpression.

In the context of the present invention, a "recombinant" or "genetically modified" microor-ganism shall be a microorganism into which an expressible protein, such as the P01) signal transduction protein variant as described herein, has been introduced.

A recombinant protein is "expressible", if it has been recombinantly provided to a microor- ganism in a way that allows for the expression of said protein by said microorganism. Usu-ally, this is achieved by transforming a microorganism with a genetic expression construct comprising the gene encoding said protein to be expressed, together with additional genetic elements, such as promoter and/or terminator sequences, that control expression of said pro-tein in said microorganism. Preferably, the protein to be expressed is transformed using an expression vector, and respective vector constructs are well known to the person of skill and dcscribcd in thc litcrature. Furthermorc, said cxpression vcctor prefcrably can causc an in- creased expression, such as, for example, overexpression of said P(II) signal transduction pro-tein in the cell. Then, said microorganism preferably expresses more than 10%, preferably more than 25% and most preferred more than 50% more of said P(ll) signal transduction pro-tein, compared to a non-increased expression.

Preferred is a method according to the present invention, wherein said microorganism is a bacterium that naturally contains a P11-regulated NAGK protein, preferably selected from Cyanohacteria, such as Aphanocapsa, Synechococcus, Svnechocvstis, Anahaena, and Spirulina. Particularly preferred are Svnechococcus elongates, Anabaena sp. strain PCC7I2O, and Anahaena variabilts.

Further preferred is a method according to the present invention, wherein said microorganism is a microorganism that does not naturally contain a P11-regulated NAGK protein, and wherein said microorganism has been geneticaHy modified to contain an expressiNe eyano-bacterial NAGK gene (argB), and optionally an expressible cyanophyein synthetase (cphA) gene and, cyanophycin depolymerase (cphB) gene and/or an expressible cyanophycin hy-drolase (cphE) gene and/or an expressible cyanophycinase (ephl) gene, preferably selected from Acinetobacter, and Desi1fltohacteriuen. The genes for the NAGK protein (argB gene product) can be preferably selected from the same organisms as indicated above for the P(II) signal transduction protein.

The activation of the enzyme NAGK as the key enzyme of the arginine biosynthesis is only found in eyanobaeteria (and plants). In all heterotrophie bacteria, such as Acinetohacter or Desufitobacterluin, NAGK is not controlled by P11. Therefore, according to the invention, these bacteria are recombinantly provided with a cyanobacterial gene for NAGK (the gene argB), in order to allow for a contr& thereof by the superactive P11-protein as provided as well.

In general, any suitable nitrogen source or combination of nitrogen sources can be used in the context of the present invention. Preferred is a method according to the present invention, wherein said nitrogen source is selected from arginine, aspartate, protamylasse, plant biomass, manure, ammonia, nitrate, atmospheric nitrogen, and mixtures thereof Particularly preferred are waste materials as nitrogen source. In one particularly preferred embodiment, the source is atmospheric nitrogen.

Further preferred is a method according to the present invention, wherein said microorganism is selected from a diazotrophic microorganism that is able to fix atmospheric nitrogen, such as, for example, heterocyst-forming cyanohacteria, such as Anahaena; non-heterocystous cyanobacteria such as Cyanothecae and heterotrophic nitrogen-fixing bacteria, such as RJüzo- h/a; Prank/a; and Azotohacteraceae. In one particularly preferred embodiment, said microor-ganism is Anahaena.

Even further preferred is a method according to the present invention, wherein said microor- ganism has been genetically modified to contain at least one genetically modified gene in-volved in the regulation of nitrogen fixation, and/or has been genetically modified to contain at least one expressible, optionally genetically modified, gene involved in nitrogen fixation, such as, for example, glutamine synthetase, wherein said genetic modification(s) increase nitrogen fixation in said microorganism. In this embodiment, the microorganisms are geneti- cally modified in order to improve the amount of the available nitrogen through nitrogen fixa-tion. This creates microorganisms that are able to more efficiently produce cyanophycin based at least in part on, for example, atmospheric nitrogen.

The genes as introduced into said microorganisms can be autologous or heterologous, as long as they can functionally interact in the pathways as required for the synthesis of cyanophyein and/or the overproduction thereof, and in nitrogen fixation. Typical heterologous genes en-code for proteins that are at least 80% identical in their amino acid sequence to an autologous protein, more preferred to at least 90%, and most preferred to at least 95%.

In one particularly preferred embodiment of the method according to the present invention, said P(II) signal transduction protein variant that is the superactive NAGK protein binder comprises a defect in 2-OG binding, and is preferably selected from the protein PUT) variant from Synechococcus elongatus PCC 7942 186N, and 186T.

In another embodiment of the method according to the present invention, the microorganism as used produces more cyanophycin (e.g. a higher amount of(wt/wt) of cellular dry matter), when compared to a microorganism carrying an unmodified P(l I) signal transduction protein.

More preferably, said microorganism produces more than 10%, preferably more than 25% and most preferred more than 50% more cyanophycin.

In another cmbodiment of the method according to the present invention, the mcthod pro-duces cyanophycin of a high quality as indicated by a molecular weight of about more than 40 kDa, preferably more than about 60 kDa, and a maximum at about 100 to 170 kDa. The cyanophycin as produced according to, for example, Steinbuchel et al. in E. coil is smaller and has a size of about 25-30 kDa. About shall mean a deviation of +1-10%.

Yet another aspect of the present invention then relates to a recombinant strain of a microor-ganism that naturally contains a NAGK protein, preferably selected from Ciyanobacteria, such as Aphanocapsa, Synechococcus, Synechocystis, Anahaena, and Spiruhna, and preferably Anahaena, comprising a P01) signal transduction protein variant that is a superactive NAGK protein binder, preferably selected from the protein PUT) variant from Synechococcus elonga-tus PCC 7942 186N, and 186T.

Yet another aspect of the present invention then relates to a recombinant strain of a microor-ganism selected from a diazotrophic microorganism able to fix atmospheric nitrogen, such as, for example, heterocyst-forming cyanobacteria, such as Anabaena; non-heterocystous cyano-bacteria such as Cyanothecae and hctcrotrophic nitrogen-fixing bacteria, such as Rhizohia; Fran/cia; and Azotohacteraceae, comprising a PUT) signal transduction protcin variant that is a superactive NAGK protein binder, which is preferably selected from the protein P01) variant from Synechococcus eiongatus PCC 7942 l86N, and l86T.

Due to their recombinant elements, these strains are of particular use as tools in the methods of the present invention. These recombinant strains according to the present invention can be genetically modified to contain at east one genetically modified gene inv&ved in the regula- tion of nitrogen fixation, and can be further genetically modified to contain at least one ex-pressible, optionally genetically modified, gene involved in nitrogen fixation, such as, for example, glutamine synthetase, wherein said genetic modification(s) increase nitrogen fixa-tion in said microorganism. In this embodiment, the microorganisms are genetically modified -10 - in order to improve the amount of the available nitrogen through nitrogen fixation. This cre-ates microorganisms that are able to more efficiently produce cyanophycin based at least in part on, for example, atmospheric nitrogen.

Yet another aspect of the present invention then relates to the use of a recombinant bacterial strain according to the present invention for the production of cyanophycin, preferably using atmospheric nitrogen as the nitrogen source or as one of the nitrogen sources. In one particu-larly preferred embodiment, said recombinant bacterial strain is derived from Anahaena.

The present invention shall now be explained further in the following non-limiting examples, with reference to the accompanying figures. For the purposes of the present invention, all ref-crcnccs as cited herein are incorporated by reference in their entireties.

Figure 1 shows microscopic pictures of the strain comprising the P(II) signal transduction protein variant that is a superaetive NACK protein binder from Synechococcus elongatus PCC 7942 186T. The recombinant strain (left) contains granulae of cyanophyein, the wild type (right) does not contain visible cyanophycin particles.

Figure 2 shows an SDS gel of extracted cyanophycin after Coomassie-staining. Due to differ-ent lengths of the chains as produced, the cyanophycin polymer does not show as a single band on the gel. The average chain length appears to be long, and thus the cyanophyein as produced is of a higher quality than the one according to Steinbuchel et al. in E.eoli (at about 25-30 kDa, see above). The cyanophycin as produced is mostly larger than 40 kDa, with a Maximum at 100 -170 kDa. Lane I shows a molecular weight marker, with 170, 130, 100, 70, 55, 40, 30, 25, 15, 10 kDa (from top to bottom). Lane 2 shows an extraction from wild type-cells, with no apparent eyanophycin. Lane 3 shows the eyanophycin extraction from the 186N strain.

Examples

186N Variant of the P11 protein In experiments as performed in the context of the present invention, the 186N variant of the P11 protein was characterized further. It was found that the 186N variant of the PIT protein was a superaetive interacting partner of NAGK (Fokina et at, 2010). This variant of the P11 pro- tein was the result of experiments with random mutagenesis (Fokina et al., 2010), and isoleu-cine (I) was replace by asparagine (N) at position 86.

1. Bacterial Strains i'nechoQ'stis sp. PCC 6803 APII comp. WT 7942 -P11 deficient, complemented with wild type P11 from Synechococcus PCC 7942 Synechocystis sp. PCC 6803 APII comp. 186N 7942 P11 deficient, complemented with 186N P11 from Svnechococcus PCC 7942 Synechocystis sp. PCC 6803 APII comp. R45A 7942 P11 deficient, complemented with R45A P11 from Svnec/iococcus PCC 7942 2. IPlasinids pJETI.2/blunt Cloning Vektor (Fermentas) pVZ 322 (Zinchenko etaL (1999)) 3. Buffer for cyanophycin-extraction Lysis-buffer Component Concentration Tris-FICI (pH 7.4) 25 mM KCI 50 mlvi MgC12 5 mlvi EDTA 0.5mM Benzamidine 1 mlvi 4. Media for the growth of Synechocystis-strains BO-Il medium (Rippka, 1988) Component Concentration NaNO3 17.6mM K2HPO4x3H2O 0.175mM MgSO4 x 71120 0.3 mM CaCI2 x 21120 0.25 mM C61-1807 0.028mM FeC5O5H7 0.028mM EDTA 0.00345 mM Na2CO3 0.375 mM Trace element solution 1 mM Additive (sterile filtered) Component Concentration NaHCO3 5mM Added to the medium directly before inoculation -12 -The BG-1 1° medium (combined nitrogen free medium) corresponds to BGI I medium (see above) except that NaNO3 is removed and substituted by an equimolar amount of NaCI.

5. Construction of the g/nB-insert The variants of the ginB-gene as used were derived from the cyanobacterium Synechococcus elongatus PCC 7942. For this, an unmodified ginS-gene, the wild type-form was used, and frirthermore, two mutated ginB-genes were used, each carrying point mutation. The 186N-mutant of the ginB-gene encoded for a PIT protein having an amino acid-exchange at position 86. In the protein, an isoleucin was replaced by an asparagine. The R45A-mulant of the ginS-gene encoded for a PIT protein having an amino acid-exchange at position 45. In the protein, an argininc was rcplaced by an alaninc.

In order to safely express the gins-gene from a foreign organism in the APII mutant of Svnechocvstis jsp. PCC 6803, using flision-PCR a construct of the respective glnB-gene from Synechococcus c/on gatus PCC 7942 and the ginB-promoter sequence from Synechocystis.sp.

PCC 6803 had to be produced. The sequences of the ginB-gene variants and the ginB-promoter sequence as required for the fusion-PCR were amplified as described herein. The ginB-insert was first ligated with the pJET 1.2/blunt cloning vector (Fermentas), and multi-plied in E. coil. Then, using enzymatic digestion, the respective ginB-construct was cut from the pJETI.2 vector, and was purified. Then, the purified glnB-construet was cloned into di-gested pVZ322-veetor (Zinehenko et at, 1999), and the pVZ322-vector with insert was then transformed into E. coil. After propagation, the plasmid was isolated from the cells, and, after butanol precipitation, was transferred by means of electroporation into the APIT mutant of the cyanobacterium Svnechocystis sp. PCC 6803.

6. Generation of the glnB-insert In order to produce the ginB-inserts consisting of the ginB-promoter sequence of Synechocys-tis sp. PCC 6803 and the respective variants of the gins-gene from Synechococcus eiongatus PCC 7942, first, the promoter region as well as the ginS-sequences were amplified in separate PCR-reactions. The three variants of the ginS-sequence were amplified from the plasmid pASK-IBA3. The sequence of the ginS-promoter was amplified from the genomie DNA of Svnechocvstis sp. PCC 6803.

-13 -7. PCR g to 50 g of genomic DNA or 0.02 jg plasmid-DNA was used as template for the PCR reactions. For the fusion-PCR, 0.02 tg of each of the templates to be fused were used. A regu-lar PCR-reaetion (100 tl) contained 0.4 pmol4d 3'-primer and 0.4 pmol4tl 5'-primer, 0.2 mM dNTP's and 2.5 U taq-DNA-polymerase (in the RedTaq master-mix 2 X from Genaxxon bio-science). For the fusion-PCR, a PCR-reaction contained 0.4 pmo1/d 3'-primer and 0.4 pmoLpi 5'-primer, 0.2 mM dNTP's, 2,5 U pJii-DNA-polymerase, and 5 jJ 10 x Pfu buffer with MgSO4 (Fermentas).

PCR was performed in the Thermocycler LabCycler from Sensoquest. The PCR program as used involved an initiation step at 98°C for 10 sec, a denaturation step at 98°C for 1 sec, a hybridization step at 55.3 °C for 9 sec, a synthesis step at 72°C for 15 see, and a final synthe-sis step at 72 °C for 1 minute. The steps of denaturation, hybridization and synthesis were repeated for 20 times.

7.1 Fusion-PCR Using fusion-PCR, both the gJnB-DNA-fragment from Synecliococcus elongatus PCC 7942 and the sequence of the ginS-promoter from the genome of Synechocystis sp. PCC 6803 were linked. In order to achieve a linking of these two DNA-fragments, and a subsequent amplifi-cation of a joint fragment of the promoter sequence of Synechocystis sp. PCC 6803 with the ginB-sequence of Svnechococcus elongatus PCC 7942, an overlapping sequence of 18 bp that was complementary to the sequence ofp68O3R was attached to ginB7942F. For the reaction, 0.02 tg of both components were used.

8. Preparation of electro-competent cells of Synechocysfis sp. PCC 6803 Although Svnechocystis sp. PCC 6803 is naturally competent, the transformation of large plasmids is inefficient. Transformation frequency can be increased by electroporation. To this end, the interfering salts had to be removed by washing a cell culture with an 0D750 of 0.8 for three times in 1 mM HEPES (pH 7.5).

9. Transformation of Synecliocystis sp. pcc 6803 using electroporation For transformation the cells as prepared above were resuspended in 100 p1 1 mlvi HEPES (pH 7.5). 60 p1 of this suspension were then mixed with 5 tg of the plasmid-DNA to be trans-formed, dissolved in 10 pi 1-120. For the electroporation the Gene PulserTM (Bio Rad) was set -14 -to 600 Ohm, 25 RF and 2 V. The length of the pulse was 13 ms. The cuvettes as used for the electroporation were held on ice for at least 20 mm before use. Immediately following the electric shock, I ml of BG-l I liquid medium without antibiotics added to each of the prepara- tions of transformcd cells. This suspension was then mixed with 50 ml of BG-1 1 liquid me-dium without antibiotics, and was incubated in 100 ml Erlenmeyer-flasks for 5 days at 30°C with shaking (120 rpm). During the incubation period, the cells were neither subjected to strong light, nor to antibiotics. The culture was then harvested and resuspended in 500 tl BG- 11 liquid medium without antibiotics. The suspension was mixed with 50 ml of BG-1 I soft-agar (BG-1 1 liquid medium with 0.75 % agar) without antibiotics. Dilutions of i0 of the cells were each plated on BG-1 I plates with gcntamycin, and incubated for additional 14 days at 30°C and white light.

10. Growth of Cyanobacteria The strains of Synechocvsth sp. PCC 6803 were grown in BG-1 I medium (see above) sup-plemented with the antibiotics as required. The cells were grown in an Erlcnmeyer flask at 30°C and white light. The cultures were shaken at 120 rpm. For larger volumes (above 500 ml), the cultures were grown in 11 glass culture flasks under aeration with pressurized air.

Proteins were isolated using common methods (Cracking of cells and centrifugation, see be-low).

11. Overexpression of proteins in E. coil 11.1 Overexpression of NAGK-His in E. coli Die ovcrexpression in E. co/i cells was performed as described in Maheswaran et al., 2004.

The BL-2 I cells of E. co/i wcrc transformed using electroporation (see above) and grown in LB-liquid medium with ampicillin. For overexpression, a culture volume of 500 ml was used.

The cells were incubated up to an 0D600 of 0.8 at 37°C with shaking at 140rpm. Before in-duction, a sample of 1 ml was taken, spun down at 5000 rpm, the supernatant discarded, and frozen at -20°C. Induction was achieved with 1 mIVI IPTU. Overexpression took place at ft (2 1°C) with shaking at 140 rpm over night. Then, the cells were harvested. Furthermore, an- other test sample was taken at 0D600 of 0.8, and treated as before induction. Both test sam-ples were analyzed using denaturating polyacrylamide gel-electrophoresis.

11.2 Overexpression of P11-strep in E. coil -15 - Die overexpression was performed as described in Maheswaran et cii., 2004. Since RB9060-cells of E. coil were used for the transformation, transformation was achieved using a heat shock method. The cells were grown as above in LB-liquid medium with ampicillin. For the overexpression, a culture volume of 500 ml was used. The cells were incubated at 37°C up to an ODÔOO of 0.8 with shaking at 140 rpm. Before the induction with AHT, a test sample of 1 ml was taken, and treated as described above. Overexpression took place at 21°C with shak-ing at 140 rpm until an 0D600 of 1.6 was reached. Then, the cells were harvested.

12. Extraction of cyanophycin from strains of Synechocystis sp. PCC 6803 The cyanobaeterium Synechocystis sp. PCC 6803 is able to synthesize cyanophycin as nitro-gen storage-compound, and to store it in specific insoluble granules (inclusion bodies).

12.1 Extraction of cyanophycin from ammonia-grown cultures using gradient centrifu-gation Cells of Synechocystis.sp. PCC 6803 were grown in 500 ml BG-1 1° liquid medium supple-mented with 5 mM NH4CI at 28°C in shaking flasks with illumination from white fluorescent lamps (at a photon flux density of 40 Rmol photons s1 m2), until they reached an 0D750 of 0.8. In this experiment, all transformants of the APTI mutant of.Snechocystis sp. PCC 6803 were treated identically. Thereby, the non-transformed APII mutant served as a negative con-trol, and the wild type Synechocystis PCC 6803 served as a positive control. Then, the strains that were transformed with the three different ginB-genes from Synechococcus eiongatus PCC 7942, the APII mutant of Svnec/iocystis sp. PCC 6803 (APII Synechocystis sp. PCC 6803 / WT7942, APII Synechocyctis sp. PCC 6803 / 186N7942 and APII Synechocystis sp. PCC 6803 / R45A7942) could be compared with thc positive control and thc ncgativc control.

The cells were harvested in a Beckman Coulter Avanti J-26XP (Beckman Coulter GmbH, Krefeld). The pellet as obtained was resuspended in 5 ml lysis-buffer and subsequently cracked open in the Ribolyser. After incubation of the suspension for 5 mm on ice, in order to allow the glass beads to settle, the supematant was pelleted by means of centrifugation in the Beckman Coulter Avanti J-26XP (Beckman Coulter GmbH, Krefeld) at 6000 x g and 4 °C for 1 hour. The pellet as obtained was resuspended in 5 ml lysis-buffer, and loaded onto 90 % Percoll (Biochrom AG, Berlin) in 0.15 M NaCI (20 ml). The density gradient centrifugation was performed in a Beckman Coulter Avanti J-26XP (Beckman Coulter GmbH, Krefeld) at 13000 x g and 4 °C for 55 mm. Then, the pellet and the bottom fraction were carefully taken up and washed in a ratio of 1:10 with lysis-buffet Following an additional centrifligation at 13000 x g and 4°C for 20 mm, the pellet as obtained was taken up and dissolved in 2 ml 0.1 M HCI. In this step, the cyanophycin stayed in solution, while most of the other proteins pre-cipitated. The suspension was pelleted in the Eppcndorf Centrifuge 5417 C (Eppendorf AG, Hamburg) for 10 mm at ft with 20800 x g. The supematant was taken up, and neutralized with 1 M ItOH (in 50 mM Tris-HC1 pH 8.0), so that the dissolved cyanophycin precipitated again. Then, the supernatant was incubated over night at 4°C. Following another centrifuga-tion in the Eppendorf Centrifuge 5417 C (Eppendorf AG, Hamburg) for 10 miii at ft with 20800 x g the pellet could be precipitated again as described above. In doing so, the purity of the cyanophycin was improved. Finally, the pcllet so obtained was dried in an exsiccatoit The dried pellet was taken up in 500 p.1 0.1 M HCI, and dissolved. Then, the protein concentration was determined using a Bradford-assay. In order to determine thc purity of the extracted cyanophycun, 1 gg total protein was loaded on a 15 % denaturating polyacrylamide gel (Laemmli, 1970), and stained.

7.3 In vilro activity test: Determination of NAGK-actlvlty using a coupled enzymatic test Using the coupled enzymatic test, isolated enzymes were tested for their enzymatic activity.

Tn this experiment, the protein activity of variants of the NAG-kinase (N-acetyiglutamate kinase) was measured alone, and in the context with different variants of P11 proteins. Fur-thermore, the activity was determined in the presence and absence of different mediators hi viny. In order to detect the enzymatic activity the oxidation of NADH to NAD+ was meas-tired photometrically at an optical density of 340 nm (0D340) for a period of 600 sec.

The analysis of the results was performed using thc software program GraphPad Piism®4 (GrapbPad Software Inc., La Jolla, CA, USA) whereby the coefficient for the conversion of the absorption as measured into units/mg was set at 48.559.

Using one molecule of ATP, the N-acetylglutamate as employed in the assay was phosphory-lated to N-acetylglutamate-phosphate by the N-acetylglutamate kinase (NAGK). The ADP as generated was regenerated to ATP by the pyruvate kinase through the cleavage of a phosphate residue from phosphoenolpyruvate (PEP). The oxidation of one molecule NADH to NAD+ by the lactate dehydrogenase reduced the pynwate as generated to lactate, the final product.

-17 -The P11 proteins as used in the experiment were the wild type-form (PIIWT), the 186N-mutant (P11 186N), and the R45A-mutant (P11 R45A). The 186N-mutant was a so-called "super-binder" that could no longer dissociate from the wild type-form of NAGK (Fokina et at, 2010).

The R45A-mutant of the P11 protein exhibited oniy a low capability to interact with the wild type-form of the NAG-kinase. No interaction was found with the 233A-mutant of the NAG-kinase (Llácer et al., 2007). For this reason, it was used in the experiment as negative control.

The wild type-form was used as a positive control of the experiment, since it was able to in-teract with both forms of the NAG-kinase (Fokina et at, 2010).

7.4 Coupled enzymatic test in the absence of P11 A reaction mix having a volume of 1 ml contained 50 mM imidazole (pH 7.5), 50 mlvi KC1, mM MgCI2, 0.4 mM NADH, 1 mM PEP, 10 mM ATP, 0,5 mM DII, 10.92 U lactate-dehydrogenase, 15.33 U pyruvate-kinase, 50 niN'l N-aeetylglutamate (NAG), and 3 tg of each of the NAG -kinase.

Both variants of the NAG-Idnase, that is, the wild type (NAGK WT) and the mutant (NAGK 322), were tested for their enzymatic activity in the absence of P11 proteins.

7.5 Coupled enzymatic test in the presence of P11 For the coupled enzymatic test in the presence of P11 proteins, 1.2 pg of each of the respective variant of the isolated PIT protein were added to the reaction mixture. Both the wild type N-acetylglutamate kinase (NAGK WI) and the mutant of the N-acetylglutamate kinase (NAGK 322) were tested for their enzymatic activity in the presence of the individual variants of the P11 protein (Fl I-WT, Fl I-186N, and P1 I-R45A) (Fokina et at, 2010).

8. Growth of Anabaena p. Anabaena sp. strain PCC 7120 (Anabaena) and derived mutant strains were grown in liquid medium according to Allen & Arnon (1955) under photoautotrophie conditions as described previously (Fiedler et al., 1998b). Routinely, Anabaena strains were grown in Erlenmeyer flasks with constant shaking. Experimental liquid cultures were grown in 750 ml bottles, bub-bled with 2% C02-enriched air. During nitrogen step-down experiments, liquid cultures were washed three times in medium without combined nitrogen and resuspended in the same me- dium to induce heterocyst formation. Mutant strains were grown with the antibiotic as re-quired.

9. Transfer of plasnilds Into Anabaena sp.

Plasmid transfer into Anabaena sp. PCC 7120 is carried out by conjugation with E. coli donor and helper cells (triparental mating). To this end, the conjugative plasnild RP-4 and helper plasmid pRL528 arc used according to published methods (Elhai & Wolk, I 988b; Wolk et al., 1984).

10. Nltrogenase activIty measurements.

Nitrogenase activity was measured as described by Valladares et al. (2007).

Claims (15)

1. Claims A method for producing cyanophycin in a recombinant microorganism, comprising a) providing a recombinant microorganism, b) providing a nitrogen source for said recombinant microorganism, and c) conversion of said nitrogen source into cyanophycin by said recombinant microor-ganism, wherein said recombinant microorganism expresses a recombinant P(II) signal trans-duction protein variant that is a sup eractive N-Acetyl-L-Glutamate (NAGK) protein bindcr.
2. 2. The method according to claim I, wherein said microorganism is a bacterium that naturally contains a P11-regulated NAGK protein, preferably selected from Cyanobac-teria, such as Aphanocapsa, Synechococcus, SynechocystLc, Anabaena, and Spirulina.
3. 3. The method according to claim I, wherein said microorganism is a microorganism that does not naturally contain PIE-regulated NACIK protein, and wherein said microorgan-ism has been genetically modified to contain an expressible cyanobacterial NAUK gene (argB), and optionally an expressible cyanophycin synthetase (cphA) gene and, cyanophycin depolymerase (cphB) gene and/or an expressible cyanophycin hydrolasc (cphE) gcnc and/or an cxprcssiblc cyanophycinasc (cphl) gene, prcfcrably selected from Acinetobacter, and Desufitobacterium.
4. 4. The method according to claim I, wherein said nitrogen source is selected from argin- me, aspartate, pmtamylassc, plant biomass, manure, ammonia, nitrate, atmospheric ni-trogen, and mixtures thereof.
5. 5. The method according to claim I, wherein said microorganism is selected from a di- azotrophic microorganism able to fix atmospheric nitrogen, such as, for example, het-erocyst-fbrming cyanobacteria, such as Anabaena; non-heterocystous cyanobacteria such as Cyanothecae and heterotrophic nitrogen-fixing bacteria, such as Rhizobia; Frank/a; and Azotobacteraceae.

   -20 -
6. 6. The method according to claim 5, wherein said microorganism has been genetically modified to contain at least one genetically modified gene involved in the regulation of nitrogen fixation, and/or has been genetically modified to contain at least one ex-pressible, optionally genetically modified, gene involved in nitrogen fixation, such as, for example, glutamine synthetase, wherein said genetic modification(s) increase ni-trogen fixation in said microorganism.
7. 7. The method according to claim 6, wherein said gene involved in nitrogen fixation is autologous or hctcrologous for said bacterium.
8. 8. Thc mcthod according to any of claims Ito 6, whcrcin said P(II) signal transduction protein variant that is the superactive NAGK protein binder comprises a defect in 2- OG binding, and is preferably selected from the protein P(Il) variant from Svnecho-COCCI'S elongatus PCC 7942 186N, and 186T.
9. 9. The method according to any of claims Ito 8, wherein said microorganism produces more cyanophycin when compared to a microorganism carrying an unmodified P(I1) signal transduction protein.
10. 10. The method according to claim 9, wherein said microorganism produces more than 10%, prcferably more than 25% and most preferred morc than 50% more cyanophy-cm.
11. 11. The method according to any of claims Ito 10, wherein the cyanophycin as produced has a molecular weight of about more than 40 kDa, preferably more than about 60 kDa, and a maximum at about 100 to 170 kDa.
12. 12. A recombinant strain of a microorganism that naturally contains NAGK protein, pref- erably selected from Cyanobacteria, such as Aphanocapsa, Synechococcus, Synecho-cvstis, Anahaena, and Spirulina, and preferably Anahaena, comprising a P(II) signal transduction protein variant that is a superactive NAGK protein binder, which is pref-erably selected from the protein P(II) variant from Synechococcus elongatus PCC 7942 186N, and 186T. -21 -
13. 13. A recombinant strain of a microorganism selected from a diazotrophic microorganism able to fix atmospheric nitrogen, such as, for example, heterocyst-forming tyanobacte- na, such as Anabaena; non-heterocystous cyanobacteria such as Cyanothecae and hot-erotrophic nitrogen-fixing bacteria, such as Rhizobia; Frankia; and Azotobacteraceae, comprising a P(ll) signal transduction protein variant that is a superactive NAGK pro- tein binder, which is preferably selected from the protein P(II) variant from Synecho-coccus elongatus PCC 7942 186N, and 186T.
14. 14. The recombinant strain according to claim 13, wherein said microorganism has been genetically modified to contain at least one genetically modified gene involved in the rcgulation of nitrogen fixation, and/or has beat genetically modified to contain at least one expressible, optionally genetically modified, gene involved in nitrogen fixation, such as, for example, glutamine synthetase, wherein said genetic modification(s) in-crease nitrogen fixation in said microorganism.
15. 15. Use of a recombinant bacterial strain according to any of claims 12 to 14 fbr the pro-duction of cyanophycin, preferably using atmospheric nitrogen as the nitrogen source or one of the nitrogen sources.