NO335366B1 - Mutant strain Pf201 of Pseudomonas fluorescence and its use in alginate production, as well as process for its preparation. - Google Patents

Abstract

2005-04-08 S a m m e n d r a g Biologically pure bacterial cultures of mutant strains of Pseudomonas fluorescens, which produce large amounts of alginate, have been described. The alginate may contain a certain amount of mannuronate and guluronate residues, possible presence and a certain level of acetyl groups in the alginate, and a desired molecular weight of the alginate. High-performance mutants with regulation of alginate production are also described. The invention further provides methods for the production of novel mutant strains of Pseudomonas fluorescens and variants thereof, and the use of the resulting strains in alginate production.

Description

Field of the invention.

This invention relates to new mutant strains Pf201 of Pseudomonas fluorescens and their use in alginate production, as well as methods for

manufacture thereof. The strain is capable of producing large amounts of alginate. The alginate is not only produced in large quantities, but also with a certain specific content of mannuronate and gulurona residues, possible presence and a certain level of acetyl groups in the alginate, and a desired molecular weight of the alginate. High-yielding mutants with regulation of alginate production have also been described.

Description of prior art.

Several microorganisms are known to produce alginate, and the most studied of the bacteria is the bacterium Pseudomonas aeruginosa. However, it is of limited use when it comes to the production of alginates for use in foodstuffs, or pharmaceutical preparations, because it is associated with primary and secondary infections in mammals or humans. Other species, which may be safer sources, usually do not produce significant amounts of alginates, or alginates with a sufficiently high molecular weight, and for this reason cannot be used.

Non-pathogenic species of Pseudomonas such as P. putida, P. mendocina and P. fluorescens are known to produce exopolysaccharides similar to acetylated alginates, Govan J.R.W. et al., J. of General Microbiology (1981), 125, pp. 217-220. Also Conti, E. et al., Microbiology (1994), 140, pp. 1125-1132, describes the production of alginates from P. fluorescens and P. putida. However, no stable excess producers of alginate are known among these strains.

US Patent No. 4,490,467, belonging to Kelco Biospecialties Ltd., describes polysaccharide production using new strains of Pseudomonas mendocina. The strains produce a good yield of the desired polysaccharide, and are relatively stable during continuous fermentation. The strains are produced by exposing a wild-type culture of P. mendocina to carbenicillin, and mutagenizing the selected, resistant, mucoid clones with a mutagenic agent. The most stable, and thus most preferred, is deposited with No. NCIB 11687. High concentrations of alginate, approximately 20 g/l, were obtained in nitrogen-limited, continuous culture with a minimal glucose medium. An alginate lyase activity was present in the cultures and resulted in a low molecular weight, low viscosity polymer with rheology similar to printing grade alginate. The degradation by the lyase enzyme was aided by the addition of proteolytic enzyme to the medium, Hacking A.J., et al., (1983), J. Gen. Microbiol., 129, pp. 3473-3480. After ten generations in continuous culture, non-mucoid variants appeared, Sengha S. S., et al., (1989), J. Gen. Microbiol., 135, pp. 795-804, page 799, second paragraph.

MOREA et al; Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene duster of Pseudomonas fluorescens; Gene, vol, 278, no. 1-2, 2001, p. 107-114, ISSN0378-1119 describes only the characterization of a limited part of the alginate biosynthetic gene cluster, i.e. it only confirms the presence of algA and algG and suggests that AlgE and /1/gX-like sequences are present in the organism. The entire alginate operon in P. fluorescens was not determined and characterized prior to the present invention.

GIMMESTAD ET AL; The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5 epimerase activity, is needed for alginate polymer formation; J. BACT., VOL. 185, NO.2, 2003, p. 3515-3523, ISSN 0021-9193 does not describe the oral stem according to the present invention.

An epimerase-negative mutant of the opportunistic pathogen P. aeruginosa was reported by Chitnis et al. (1990), J. Bacteriol., 172, pp. 2894-2900. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants unable to incorporate guluronic acid (G) residues into alginate were independently isolated. Analyzes using G-specific alginate lyase and <1>H nuclear magnetic resonance analyzes showed that G residues were not present in the alginates secreted by these mutants. Gold-berg and Ohman, 1987, J. Bacteriol., 169, pp. 1593-1602, produced up to 1.7 g/l alginate from FRD1 in shake flasks. As usual for spontaneous alginate producers, non-mucoid revertants ("revertants") occurred frequently (Flynn and Ohman, 1988, J. Bacteriol., 170, pp. 1452-1460).

There is therefore still a need on the market for suitable sources for reliable alginate production in large quantities. In particular, there is a need for stable sources that produce large quantities of high-quality alginate with a defined structure and desired molecular weight, and especially for a source for the production of large quantities of biologically active alginate. Furthermore, there is also a need for the production of pure mannuronan, which can be subjected to in wfro-epimerization to obtain alginates with a predetermined guluronate residue (G) content.

Summary of the invention.

The present invention provides new mutant strains of P. fluorescens, which are stable and produce large amounts of alginate. Some embodiments of the invention include mutants thereof, which produce alginates with a defined structure with regard to the content of mannuronate and gulurona residues, a possible presence and specific level of O-acetyl groups, and a desired molecular weight of the alginate molecules. High-yielding mutants with regulated alginate production, and methods for their production, are also described. Other aspects of the invention are: methods for the production of the new mutant strains of P. fluorescens, including mutants thereof, and the use of the resulting mutants in the production of alginates, in particular medium or large-scale fermentor production of alginates, and more particularly the production of biologically active alginates , or pure mannuronan. The resulting alginates are useful in various food and industrial products, such as foodstuffs, animal feed, cosmetics and pharmaceutical preparations; they may also constitute an intermediate suitable for further modifications with mannuronan C5 epimerases, for example with the epimerases of US Patent No. 5,939,289.

Detailed description of the invention.

The present invention provides a mutant strain of P. fluorescens, being strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof. As the said strain produces at least 10 g of alginate per 40-55 g carbon source per liter medium at least 10 g alginate per 50-55 g of carbon source per liter of medium or at least 10 g of alginate per 40 g of carbon source per liter of medium.

The present invention further comprises mutant strain of P. fluorescens according to claim 1, said mutant strain being selected from the group consisting of mutant strain Pf201 deposited as NCIMB NO. 41137, Pf2012 deposited as NCIMB NO. 41138, Pf2013 deposited as NCIMB NO. 41139, Pf20118 deposited as NCIMB NO. 41140, Pf20137 deposited as NCIMB NO. 41141, Pf20118alglJ A deposited as NCIMB NO. 41143, Pf20118algFA deposited as NCIMB NO. 41142, Pf20118AlgLH203R deposited as NCIMB NO: 41144 and Pf201MC deposited as NCIMB NO: 41145.

The present invention further comprises a mutant strain of P. fluorescens according to any one of claims 1 to 4, said mutant having the ability to produce an alginate which consists only of mannuronate residues only. Whereas said mutant has been selected from the group consisting of the mutant strains Pf2012 deposited as NCIMB 41138, Pf2013 deposited as NCIMB NO. 41139, Pf20118 deposited as NCIMB NO. 41140 and Pf20137 deposited as NCIMB 41141.

In a third aspect, the present invention comprises a mutant strain of P. fluorescens according to any one of claims 1 to 4, said mutant having the ability to produce alginate which has a defined guluronate residue (G) content of between 0 and 30%. Such embodiments can be produced using methods according to the invention, by replacing the wild-type α/gG gene with a mutant gene, or altering the α/gG gene to encode a mannuronan C-5 epimerase enzyme with lower specificity activity than the wild-type enzyme.

In a fourth aspect, the invention comprises a mutant strain of P. fluorescens according to any one of claims 1 to 4, said mutant further comprising a mutant a/gF, algl and/or algJ gene which codes for an acetylase enzyme with reduced activity or which is inactivated and thereby produces alginate without, or with a reduced number of O-acetyl groups.

The present invention further comprises a mutant strain of P. fluorescens according to claim 5 or 9, said mutant being selected from the group consisting of the mutant strains Pf20118alglJA deposited as NCIMB NO. 41143 and Pf20118algFA deposited as NCIMB NO. 41142.

Such embodiments can be produced by deleting part or all of the a/gl, a/gJ and/or a/gF genes. The mutant variant strains Pf20118alglJA and Pf20118algFA are capable of producing large amounts of an alginate without, or with a reduced number of, O-acetyl groups, and represent preferred embodiments of this aspect of the invention.

In a fifth aspect of the present invention, the pure mutant strain of P. fluorescens is capable of producing large amounts of an alginate of a desired molecular weight. The molecular weight of the alginate is preferably between 50,000 and 3,000,000 Daltons. Such embodiments can be produced by replacing wild-type algL with a mutant gene encoding an alginate lyase enzyme with lower specific activity than the wild-type lyase enzyme. The pure mutant variant strain Pf20118Algl_H203R represents a preferred embodiment of the said mutant, which is able to produce large amounts of an alginate with a desired high molecular weight.

The present invention further comprises the mutant strain of P. fluorescens according to claim 1, said mutant strain being an alginate biosynthetic operon regulated by an inducible promoter different from the naturally occurring promoter, and possibly one or more effector genes, in which said strain is obtained by a method which comprises the following steps: (i) switching the alginate biosynthetic operon promoter to a mutant strain of P.fluorescens, which is strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof, with an inducible promoter by homologous recombination, (ii) optionally introducing effector genes into the bacterium by (i) by homologous recombination, transposon mutagenesis or by means of a plasmid, (iii) culturing the bacterium produced in steps (i) and optionally step (ii) and then selection of mutants, (iv) selection from the mutants of (iii) a mutant that produces at least 10 g of alginate per liter of medium. Where the inducible promoter is a Pm promoter or mutant thereof or the inducible promoter is a Pm promoter, and furthermore the effector gene comprises xylS.

A further aspect of the invention provides a method for producing a mutant strain of P. fluorescens, comprising (a) a mutant strain of P. fluorescens, which is the strain Pf201 deposited as

NCIMB NO. 41137, is contacted with a mutagenic agent, and

(b) the treated bacterium of step (a) is grown in the presence of one or more

antibiotics, and

(c) antibiotic resistant mucoid mutants are isolated by selection, and (d) the alginate production properties of the isolated mucoid mutants in step (c)

is determined.

The mutagenic agent from step (a) of the method is preferably nitrosoguanidine, and the antibiotics used in step (b) are a β-lactam and/or aminoglycoside antibiotic, the antibiotic preferably being carbenicillin. The antibiotic may be present in the range of 800-1000 µg/ml medium, and more preferably in amounts of 900 µg/ml medium.

In yet another aspect, the present invention provides a method for producing a mutant strain of P. fluorescens, wherein (i) the alginate biosynthetic operon promoter of mutant strain of P. fluorescens, which is strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof, is exchanged for an inducible promoter by homologous recombination, and (ii) is introduced into the bacterium from (i) by homologous recombination, transposon mutagenesis or by means of a plasmid, and

(iii) mutants are grown and then isolated by selection, and

(iv) the alginate production properties of the isolated mutants in (iii) are determined, and

(v) a mutant that produces at least 10 g of alginate per liter of medium is selected.

In yet another aspect, the present invention provides methods for producing a mutant strain of P. fluorescens, wherein (a) the wild-type a/gG gene encoding the C-5 epimerase is cloned into a plasmid or minitransposon and mutagenized by chemical mutagenesis or by PCR for

to form a library of mutagenized algG genes,

(b) a derivative of an alginate-producing strain of P. fluorescens which

lacking the a/gG gene (the Aa/gG strain) is constructed,

(c) the library of mutagenized algG according to step (a) is transferred to the Aa/gG strain of P. fluorescens and the plasmid or transposon-containing strains are identified and analyzed for alginate production and epimerase activity, (d) plasmid or transposon-containing strains containing a mutant α/gG gene encoding an epimerase that provides alginate with a residual guluronic acid content between 0 and 30% are identified by the analysis in

step (c), and

(e) the mutant a/gG gene identified in step (d) is cloned into a gene replacement vector, and (f) the gene replacement vector of step (e) is then transferred into an alginate-producing strain ag P. fluorescens to replace its a/gG gene with the mutant a/gG gene, enabling it to express the mutant gene. In yet another aspect, the present invention provides a method for producing a mutant strain of P. fluorescens in which (a) one or more amino acids, which by mutagenesis and subsequent screening are identified as important for epimerization, are changed, at the gene level, by site-specific mutagenesis to amino acids different from those occurring in both the mutant and wild-type a/gG protein, and (b) the mutant gene is cloned into a gene replacement vector and this vector is transferred into an alginate-producing strain of P. fluorescens in which it replaces the wild-type a/gG gene and is able to be expressed.

In other aspects, the invention provides the use of at least one mutant strain of P. fluorescens according to any one of claims 1 to 22, for the production of alginate.

In other aspects, the invention provides the use of at least one mutant strain of P. fluorescens according to any one of claims 1 to 22, for large scale fermentor production of alginate.

In other aspects, the invention provides the use of the alginate consisting of only mannuronate residues produced by at least one mutant strain of P. fluorescens according to claim 6 or 22, in the manufacture of a food or industrial product including a pharmaceutical preparation, cosmetic, animal feed or food product, or as a intermediate for in vitro C-5 epimerization.

The mutant strains Pf201, Pf2012, Pf2013, Pf20118, Pf20137, Pf20118algFA, Pf20118alglJA, Pf20118Algl_H203R and Pf201 MC according to the invention have been deposited in The National Collections of Industrial Food and Marine Bacteria Ltd.

(NCIMB) on July 16, 2002, accompanying accession numbers: 41137, 41138, 41139, 41140, 41141, 41142, 41143, 41144, 41145 respectively. The deposits were made in accordance with the Budapest Convention.

Definitions.

The new mutant strains, and variants thereof, according to the present invention, produce alginate in large quantities, with "large quantities", as used here, meaning at least 10 g of alginate per litres. Amounts of 10 g of alginate per liter of medium is preferably obtained from 40-55 g of carbon source per liter medium, more preferably 50-55 g carbon source per liter of medium, or most preferably of 40 g of carbon source per liter medium. The alginate yield can reach 35 g of alginate per liters, but amounts of about 20% to 50%, by weight, of the carbon source used, are more frequently obtained.

Suitable "carbon sources" can be selected from, but are not limited to, monosaccharides, disaccharides, oligosaccharides, polysaccharides, alcohols, organic acids, and are, for example, fructose, glucose, galactose, sucrose, lactose, glycerol, starch, whey , molasses, sugar syrups or lactic acid (lactate), but also other C sources, as set forth in standard textbooks, such as Bergey's Manual of Systematic Bacteriology, publishers ("editors") Noel R. Krieg and John G. Holt, 1984, Baltimore , USA, can just as well be used. It should be understood that the use of carbon sources, which must not be converted to their corresponding triose phosphates, via the Entner-Doudoroff pathway before they can be used for alginate production by the mutant bacteria, will normally give the highest yields, Banerjee et al., J. Bacteriol., 1983 , pp. 238-245. Preferably, production of more than 10 g alginate/l medium by the mutant strains of P. fluorescens according to the invention is achieved if 40 g fructose or glycerol per liter of medium is used as a carbon source. The large-scale alginate production can be carried out in any suitable manner known to a person skilled in the art, but preferably takes place in a fermenter. Fermentation is batch, fed-batch or continuous, possibly with feed of carbon sources and other suitable ingredients. The fermentation is carried out at a temperature within the interval S-SS^C. Temperatures in the lower range of this range may be chosen in certain cases, but preferably the fermentation is carried out at a temperature of from 20°C to 30°C.

Selection of media, oxygenation, pH, fermentation time, agitation, and other possible conditions for the fermentation are considered to be within the general knowledge of the art, and it must be understood that an enormous number of combinations of two or more conditions can lead to the same high amount of alginate yield, and that the present invention is not limited to a specific combination of such conditions.

The mutant strains, and variants thereof, according to the present invention are "stable", i.e. they do not revert to non-alginate producing strains when cultured over 60 generations. The mutants were grown in PIA medium in shake flasks under standard culture conditions, as set forth in Materials and Methods, except that the medium was replaced with fresh PIA medium every 24 hours (successive cultures).

The "mutant strain" used herein includes mutant strains of P. fluorescens Pf201, as well as variant mutant strains, all of which produce alginate in large amounts. In preferred embodiments, "mutant strains" refers to mutant strains of P. fluorescens Pf201, all of which produce alginate in large amounts. The variants may be the result of further mutagenesis of the Pf201 mutant strain, and/or further genetic engineering, or the result of genetic engineering or mutagenesis of a wild-type P. /Zuorescens strain. Variance-

tene will produce large quantities of alginates with certain defined structures. Also variants containing any combination of the mutations defined here are considered to be covered by this term.

The alginate produced according to the invention will have a "desired molecular weight". Preferably, alginate with a molecular weight (Mw.) in the range of 50,000 to 3,000,000 Daltons, more preferably within 200,000 to 2,000,000 Daltons, and most preferably above 300,000 Daltons, is produced.

By the term "biologically active alginate", as used herein, is meant an alginate which has an effect on a biological system, i.e. certain bioactive alginate molecule structures are known to effect biological responses in certain cellular systems. Such biological alginates have a lower content of guluronic acid (guluronate) residues, from 0 to 30% of the total uronic acid content, and preferably the guluronic acid residue content is between 1% and 15%, and more preferably within 1% and 10%.

Description of the figures.

Figure 1: Restriction endonuclease map of suicide vectors pHE55 and pMG48, see Table 1. Only unique restriction enzyme sites shown. Figure 2: Growth and alginate production in fermentations with mutant strains of P. fluorescens NCIMB 10525. Figure 3:<1>H-NMR spectra of alginate produced by P. fluorescens mutant strains Pf201 and Pf20118.<1>H-NMR spectrum of mannuronan from the other epimerase-negative mutants (Table 3) were identical to that of Pf20118. Figure 4: The alginate biosynthetic operon and the upstream open reading frame from P. fluorescens are shown. The cloned fragments are marked as boxes on the map line. Only restriction sites used for cloning are shown. The total length is 18 kb. Figure 5: Restriction endonuclease map of plasmid pMC1. Only unique restriction enzymes are shown.

General description of materials and methods.

Starting materials and dwarfing media used for bacterial dwarfing.

The bacterial strains, phages and plasmids used in the present invention are listed in table 1 below. E. coli and P. fluorescence strains were routinely grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, and 5 g/l NaCl), or on an LA medium, which is LB medium containing 15 g/l agar, at 37°C, respectively 30°C. Pseudomonas Isolation agar (PIA, Difco) was also used for propagation of P. fluorescens. E. coli used for X-phage propagation was grown in LB medium supplemented with maltose (0.2%) and MgSC>4 (10 mM). Antibiotics, when used in routine culture experiments, were present at the following concentrations: ampicillin 100-200 µg/ml, kanamycin 40 µg/ml, tetracycline 12.5 µg/ml (E. coli) and 30 µg/ml (P .fluorescence).

Production of P . fluorescence - a \ Q ' food: culture media and culture conditions.

Cultivation media:

Production of alginate in shake flask experiments was carried out in PIA medium containing bacteriological peptone (20 g/l), MgCl2 (1.4 g/l), NaCl (5 g/l), K2SO4 (10 g/l) and 87 % glycerol (20 ml/l), or in PIA medium with reduced salt (PIA medium without K2SO4). The proteases (Alkalase 2.41 (0.15 ml/l) and Neutrase 0.51 (0.15 ml/l)) were added to reduce extracellular alginate lyase activity, unless otherwise indicated. Alkalase and Neutrase were purchased from Novo Nordisk.

Production of alginate in a fermenter was carried out in PM5 medium containing: fructose (40 g/l), yeast extract (12 g/l), (NH4)2SO4 (0.6 g/l), Na2HP04x2H20 (2 g/l), NaCl (11.7 g/l), MgSO 4 x 7H 2 O (0.3 g/l) and clerol FBA622 (anti-foam) (0.5 g/l). The proteases (Alkalase 2.41 (0.25 ml/l) and Neutrase 0.51 (0.25 ml/l) were added to reduce extracellular alginate lyase activity.

Preparation of standard inoculum (frozen culture with glycerol as blood preservative).

A colony from an agar plate (incubated at 30°C for 2-3 days, PIA medium) is transferred to a shaking flask (500 ml, with guide plate) containing 100 ml of LB medium. The shaking flask is incubated at 30°C for 16-20 hours in a rotary shaker (200 rpm, opening width 2.5 cm). For preservation, sterile glycerol is added to the medium to a concentration of 15%. The mixture is transferred to sterile cryo-medicine bottles (Nunc) and stored at -80°C.

Preparation of inoculum for production trials in shaking flasks and fermentor.

1 ml of standard inoculum is transferred to a shaking bottle (500 ml, with baffle plate) containing 100 ml of LB medium. The shaking flask is incubated at 30°C for 16-20 hours in a rotary shaker (200 rpm, opening width 2.5 cm).

Alginate production in a shaking flask.

1-2% by volume of inoculum (see above) is transferred to a shaking flask (500 ml, with baffle plate) containing 100 ml of PIA medium or PIA medium with reduced salt. The shaker flask is incubated at 25° for 48 hours in a rotary shaker (200 rpm, stroke width 2.5 cm).

Alginate production in a fermenter.

2-3% by volume of inoculum from the shaking bottle is transferred to a 3-litre fermentor (Applicon), with 1.4 liters of PM5 medium. The fermentations are carried out at 25°C. The pH from the start is adjusted to 7.0-7.2. The pH is adjusted at 7.0 with NaOH (2 M) and the pH control is activated when the pH reaches this value. The air flow through the culture medium is 0.25 litres/litre of medium (wm) for the first 8-10 hours, then it is gradually increased up to 0.9-1.0 vvm. The dissolved oxygen is regulated at 20% saturation by automatic regulation of the speed of the stirring device.

Standard techniques used.

Plasmid isolation, enzymatic manipulations of DNA and gel electrophoresis were performed by the methods of Sambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual (Third Edition). Cold Spring Harbor Laboratory Press. Qiaquick Gel Extraction Kit and Qiaquick PCR purification kit (Qiagen) were used for the DNA purifications from agarose gels, respectively enzymatic reactions. Transformation of E. coli was performed as described by Chung et al., 1989, Proe Nati Acad Sei USA, 86, pp. 2172-2175, or using heat shock competent rubidium chloride cells. PCR for cloning and allele identification was performed using the Ex-pand High Fidelity PCR system (Boehringer Mannheim). As templates, either plasmid DNA or 1 µl of an overnight culture of P. fluorescens was used. In the first denaturation step, the reaction mixtures were heated to 90° for three minutes to ensure both cell lysis and full denaturation of the DNA. Site-directed mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Primers listed in Table 2 were purchased from Medprobe or from MWG-Biotech AG. Nucleotides in the primers that differ from those in the wild-type sequence are written in bold, and the restriction enzyme sites are underlined. DNA sequencing was performed using a Big-Dye kit (Applied Bio-systems).

Construction of suicide vectors for use in P . fluorescence.

To achieve homologous recombination of P. fluorescens, two different suicide vectors, pHE55 and pMG48, were constructed, see Figure 1. The construction of pHE55 is described in Table 1. It is an RK2-based vector lacking the gene encoding TrfA, which is required for replication of the plasmid. It also confers resistance to ampicillin and tetracycline, which can be used to select integrants. Expression of sacB, which encodes levan-sucrase from Bacillus subtilis, has been shown to be lethal to many Gram-negative bacteria when grown on 5% sucrose (Gay et al., 1985, J. Bacteriol., 164, p .918-921). However, in strain NCIMB 10525 of P. fluorescens, cultivation of non-mucoid and tetracycline-resistant transconjugants on sucrose resulted in glassy colonies, as if the strain were using the sucrose to produce a polymer. SacB and sucrose selection could thus not be used for this strain to positively select double crossovers. pHE55 was used as a suicide vector in some experiments, where alginate production could be used as a marker.

The plasmid pMG48 was constructed as an alternative recombination vector. The sacB gene in pHE55 was replaced by a gene encoding a TrfA-LacZ fusion protein, as described in Table 1. This protein shows β-galactosidase activity, but the essential parts of TrfA are missing. When using plates containing XGal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), 60 µl of a 20 mg/ml stock solution was added to each agar plate used for screening, β-galactosidase- the activity allows blue/white screening both for integrants (blue colonies) and later for the second recombination case (white colonies).

Homologous recombination.

The DNA sequence containing the mutation of interest, either a point mutation, insertion or deletion, together with flanking DNA of at least 0.5 kb on each side, was cloned into a suicide vector, either pHE55 or pMG48. E. coli S17.1 was transformed with the plasmid of interest and the P. fluorescens strain to be mutated was incubated in LB medium overnight. They were then incubated in fresh LB medium, 1% inoculum was used. E. coli was cultured for two hours, P. fluorescens for four hours before conjugation. One ml of each culture was then mixed and centrifuged for 15 min. at 3000 rpm. Most of the supernatant was removed and the cells were resuspended in the remaining liquid. The droplets containing the cells were transferred to LA medium and incubated at 30°C overnight. Cells were removed using a sterile spatula, resuspended in LB medium, and dilutions were plated on Pseudomonas Isolation agar (PIA, Difco) with appropriate antibiotics and X-Gal when the vector allowed blue/white selection. A non-mucoid transconjugant colony of each mannuronan-producing strain was incubated in 2-6 sequential liquid cultures overnight in the absence of tetracycline, to allow loss of the integrated plasmid. Exponentially growing cultures were diluted 10<4->10<9>fold and plated on the appropriate medium for screening for the different strains.

Measurement of G content and degree of O-acetylation of the alginate using NMR spectroscopy.

Samples from fermentations were diluted in 0.2M NaCl and centrifuged to remove the bacterial cells. For preparation of samples for determination of degree of acetylation, alginate was precipitated from the cell-free supernatant by addition of one volume of isopropanol (4^), and then collected by centrifugation. The precipitated alginate was then washed twice with 70% ethanol, once in 96% ethanol, and redissolved in distilled water before further treatment. For the preparation of samples for determination of the G content, the alginate in the cell-free supernatant was deacetylated with mild alkaline treatment, as described in Ertesvåg and Skjåk-Bræk, 1999, in Methods in biotechnology 10, Carbohydrate Biotechnology Protocols, Bucke, pp. 71- 78. Humana Press Inc. deacetylated alginate was isolated from the cell-free supernatant by acid precipitation, adding HCl to pH 2. The precipitated alginate was collected by centrifugation, redissolved in distilled water, and neutralized with alkali. To reduce the viscosity of the polymer for NMR analysis, the samples were degraded by mild acid hydrolysis, to a final average degree of polymerization (DPn) of approximately 35, i.e. 35 residues in the polymer chain, neutralized and freeze-dried, Ertesvåg and Skjåk-Bræk, 199, above. NMR spectra were obtained using a Bruker 300 MHz Spectrometer. The spectra were integrated, and the fractions of gulurone residues (Fe), mannuronate block residues (Fmm) and alternating block residues (Fm<g=g>m) and degree of acetylation were calculated as described in Grasdalen, 1983, Carbohydr. Res. 118, pp. 255-260, and Skjåk-Bræk, Grasdalen and Larsen, 1986, Carbohydr. Res., 154, pp. 239-250.

Measurement of intrinsic viscosity of the alginate and direct measurement of alginate content in fermentation samples.

The alginate produced was isolated, deacetylated, acid-precipitated, redissolved and neutralized as described above. The neutralized alginate solution was added with isopropanol to precipitate the alginate a second time. The precipitated alginate was washed twice with ethanol (first 70% and then 96% ethanol), redissolved in distilled water and dialyzed against distilled water for 48 hours. After dialysis, the sample was freeze-dried and weighed. The intrinsic viscosity of the alginates was determined with a Scott-Geråte apparatus with automatic dilution, using an Ubbelodhe capillary (<J>=0.53 mm) at 20° and an added salt concentration of 0.1 M NaCl. The principle of the procedure is described in Haug and Smidsrød, 1962, Acta. Chem. Scand., 16, pp. 1569-1578.

Enzymatic determination of alginate content in fermentation samples.

Alginate content was measured using the M-specific lyase from abalone and G-lyase from Klebsiella aerogenes, as described by Østgaard, 1992, 19, Carbohydr. Polymers, pp. 51-59.

Samples from fermentations were diluted (2-20 fold) in 0.2M NaCl, centrifuged to remove bacterial cells, and deacetylated, as described above. The deacetylated samples were then diluted in buffer (Tris-HCl (50 mM), NaCl (0.25 M), pH 7.5) to a final concentration of 0.005-0.05% alginate. LF 10/60 (FMC Biopolymer AS) or mannuronan, produced and measured as described here, were used as alginate standards in the analysis. For the assay, one volume of sample, or standard and 0.06 volumes of alginate lyase solution (approximately 1 u/ml) are added to two volumes of buffer (Tris-HCl (50 mM), NaCl (0.25 M), pH 7 ,5) and incubated for 3 hours at 25°C. The absorbance at 230 nm is recorded before and after the incubation. The differences in the A230 nm values before and after incubation are used to calculate the alginate content in the sample. The results, using this assay, agree very well with the direct measurement of the alginate content described above.

Determination of lyase activity.

Bacterial cells from the fermentations were collected by centrifugation, resuspended in buffer (Tris-HCl (50 mM), NaCl (0.25M), pH 7.5), to an optical density of 3-10 at 660 nm and sonicated. The extracts after ultrasound treatment were examined for lyase activity. M-specific lyase from abalone (described by Østgaard, 1992, 19, Carbohydr. Polymers, pp. 51-59) was used as a standard. The lyase activity in the samples was determined by measuring the degradation rate of mannuronan using a Scott-Geråte Ubbelodhe (instrument no. 53620/II). Mannuronan (1 mg/ml) was dissolved in buffer (Tris-HCl (12.5 mM), NaCl (62.5 mM), pH 7.5). 4 ml of mannuronan substrate solution and 0.4 ml of diluted standard solution, or sample, were added to the Ubbelodhe capillary. The time for the solution to pass the capillary on Ubbelodhe was measured every 2 minutes over a time period of one hour. The analysis was carried out at 25°C. Based on the data from the analysis, the degradation rate of mannuronan was calculated and compared with the lyase activity in the sample. A standard curve was obtained using the abalone M-lyase as a standard (0.005-0.05 u/ml). 1 unit of lyase activity is defined as described by Ertesvåg et al., J. Bacteriol. (1998), 180, pp. 3779-3784.

Examples of the invention.

Example 1

Preparation of mutant strain Pf201.

The wild type of P. fluorescens NCIMB10525 was purchased from The National Collections of Industrial Food and Marine Bacteria Ltd. (NCIMB). The wild type does not produce significant amounts of alginate. To isolate alginate-overproducing mutants, exponentially growing cells of P. fluorescens NCIMB 10525 were subjected to nitrosoguanidine (NG) mutagenesis. The strain was grown in nutrient medium (CM67, Oxoid) with 0.5% yeast extract, and washed twice in 0.1 M citrate buffer (pH 5.5) before treating the cells with 25 µg/ml nitrosoguanidine (NG) in citrate buffer for 1 hour at 30^. The mutagenized cells were washed with 0.1 M phosphate buffer, pH 7.0, containing KH 2 PO 4 (13.6 g/l) and NaOH (-2.32 g/l) and inoculated (2%) in nutrient medium with yeast extract. The cells were grown overnight and then frozen as 1 ml aliquots of NG stock ("stock").

Dilutions of the culture were plated on PIA medium containing carbenicillin (900 µg/ml) and incubated at 30°C. A few mucoid mutants were observed. From the screening, which included inspection of more than 4<*>10<5> colonies, the two most mucoid mutants were selected for further evaluation in fermentor studies. The better mutant, Pf201, yields 11-13 g of alginate per liter of PM5 medium containing 40 g of fructose as a carbon source per litres, as shown in Figure 2. For cultivation conditions and medium composition, see Materials and Methods. The alginate produced by the Pf201 mutant using the PM5 medium containing fructose, and under standard conditions, contains approximately 30% G (guluronate residues) with a complete absence of G blocks, as can be estimated from Figure 3. Based on the unique alginate production properties, the Pf201 strain was chosen for further development of the strain. The P. fluorescens mutant Pf201 from example 1 is deposited in NCIMB, with accession no. 41137.

Example 2

Cloning and sequencing of parts of the biosynthetic alginate operon.

A gene library for the wild-type strain NCIMB 10525 was constructed in XDASH II (lambda Dash II) (purchased from Stratagene). Chromosomal DNA was isolated as

described by Ausubel et al., 1993, Current protocols in molecular biology. Greene Publishing Associates, Inc and John Wiley & Sons Inc, New York. The gene library was then constructed by inserting partially Sau3AI-digested chromosomal DNA from NCIMB 10525 into BamHI-digested lambda Dash II and infecting E. coliXL1-Blue

MRA(P2) with the in wfro-packaged subjects, according to the manufacturers' instructions (Stratagene BamHI/Gigapack III Gold Extract). Labeling of DNA probe and detection of hybridizing X clones was done using the DIG DNA Labeling and Detection Kit (Boehringer Mannheim), according to the manufacturers' instructions. A 3.8 Mfe\- Nco\ DNA fragment from pBBg10 containing algG flanked by portions of a/gE and a/gX from P. aeruginosa was labeled and used to screen the P. ffuorescens library. One hybridizing phage, designated PfX1, was detected using this system, and X DNA was isolated using the Lambda Midi Kit (QUIAGEN). The insert was subcloned as Sal/I digested DNA fragments into pGEM11, resulting in the four subclones pMG24-27. Sequencing the ends of the sub-clones and comparison with the alginate biosynthetic operon from P. aeruginosa revealed that PfX1 contains the downstream part of the alginate biosynthetic operon from the 3' part of a/gE (Figure 4).

pMG26 and pMG27 were sequenced by Quiagen Sequencing & Genomics to obtain the complete sequence of a/gGXLIJFA. This gene organization appears to be similar to previously reported biosynthetic alginate clusters in; May and Chakrabarty, 1994, Trends Microbiol., 2, pp. 151-157, Rehm et al., 1996, J. Bacteriol., 178, pp. 5884-5889, Penaloza-Vazquez et al., 1997, J Bacteriol. , 179, pp. 4464-4472, Vazquez et al., 1999, Gene, 232, pp. 217-222. The sequence has been submitted to GenBank and given the accession number AF527790.

Example 3

Preparation of epimerase-negative variant strains.

The mutant strain Pf201 from Example 1 was subjected to further mutagenesis using nitrosoguanidine using a modification of the procedure described in Example 1. Exponentially growing cells of P. fluorescens NCIMB 10525 were subjected to nitrosoguanidine (NG) mutagenesis: The bacterial cells were washed twice with an equal volume of Tris/maleic acid (TM) buffer, pH 6.0, containing NH4SO4 (1.0 g/l), CaCl2<*>2H20 (4.4 mg/l), KN03 (6.1 mg/l), maleic acid (5.8 g/l), Tris(hydroxymethyl)aminomethane (6.05 g/l), FeS04<*>7H20 (0.25 mg/l) and MgS04<*>7H20 (0 .1 g/l). The cells were resuspended in 80% of the original culture volume of TM buffer and exposed to NG (50 µg/ml) for 1 hour at 30°C. The mutagenized cells were washed with 0.1 M phosphate buffer, pH 7.0, containing KH 2 PO 4

(13.6 g/l) and NaOH (-2.32 g/l) and inoculated (2% inoculum) in LB medium and incubated overnight. The death rate in the mutagenesis procedure was calculated to be approximately 90% using a non-mutagenized aliquot of the culture as a control. After mutagenesis, the culture was grown in LB medium overnight, and dilutions of the cells plated on LA medium containing G-lyase from Klebsiella aerogenes (approximately 0.1 u/dish), as described by Chitnis. et al, 1990, J. Bacteriol., 172, pp. 2894-2900. This G-lyase cleaves only the G-M (guluronate-mannuronate residue) and G-G (guluronate-guluronate residue) bonds in the alginate, Haugen et al, 1990, Carbohydr. Res., 198, pp. 101-109.

Mucoid mutants appeared at a frequency of about 1 in 7500 on such selective plates. One mucoid mutant was isolated and designated Pf20118. Pf20118 was grown in a fermenter under standard culture conditions using PM5 medium, see Materials and Methods. The polymer produced was analyzed by<1>H-NMR spectroscopy. The results of this analysis showed that the mutant produced pure mannuronan, see Figure 3. Several fermentations were performed with Pf20118 using standard culture conditions and the PM5 medium. Volumetric yields were in the range of 14-16 g mannuronan per liter of 40 g fructose per liter of medium, in approximately 35-hour fermentations. The P. f/t/orescens mutant Pf20118, derived from Pf201, was subjected to more than 70 different fermentor trials, none of which indicated instability in the mannuronan-producing properties. Both Pf201 and Pf20118 have been cultured for 60 generations without the appearance of non-mucoid colonies. Although it seemed possible that Pf20118 had a defect in the mannuronan C-5 epimerase gene algG, it could not be ruled out that the mutations affected other proteins, which could somehow be required for epimerization. A preliminary localization of the mutations responsible for the mannuronan-producing phenotype was performed by gene replacement of the α/gG allele in each of the mutants with wild-type α/gG. A gene replacement vector, pMG31, encoding wild-type a/gG and the first 135 bp of the downstream a/gX was constructed as described in Table 1. The plasmid was conjugated into Pf20118, as described in Materials and Methods, using of PIA-containing tetracycline as selective medium. Non-mucoid colonies appeared due to the disruption of the alginate biosynthetic operon when pMG31 was recombined into algG. A non-mucoid transconjugant colony was incubated in 2-6 sequential liquid cultures overnight in the absence of tetracycline to allow loss of the integrated plasmid. Exponentially growing cultures were diluted 10<4->10<9>fold and plated on PIA agar plates to screen for mucoid revertants. Mucoid colonies were then again plated on L-agar containing G-lyase, to test whether they produced epimerized alginate. Such non-mucoid revertants were found, confirming that the mutation must be in the DNA fragment corresponding to the algGX fragment of pMG31. The a/gG gene was amplified by PCR using the primers Pfa/gG3r and Pfa/gG4f, sequenced and the mutation identified, see Table 3.

Three other epimerase-negative mutant derivative strains were prepared according to the procedure set forth above and designated Pf2012, Pf2013, and Pf20137, respectively. They all have an identified mutation in their a/gG gene, which results in a different amino acid in their AlgG gene product, as shown in Table 3 below, and this amino acid change is sufficient to inactivate the protein. The mutants produced approximately the same levels of pure mannuronan as Pf20118 when grown under the same conditions. The epimerization defect in the mutants could be reversed by recombination with the wild-type gene in pMG31. The mutant strains in Table 3 were deposited in NCIMB under the accession numbers: Pf2012 has NCIMB no. 41138, Pf2013 has NCIMB no. 41139, Pf20118 has NCIMB no. 41140 and Pf20137 have NCIMB no. 41141.

Example 4

Production of acetylase-negative and modified variant strains, Pf20118a/ qFA and Pf20118a/ qlJA.

A Pf20118 α/gF deletion mutant was first generated by constructing a mutant DNA fragment containing flanking sequences of an in-frame deletion of parts of α/gF, and then ligating the fragment into the suicide vector pMG48, as described in Table 1 The resulting plasmid, designated pMG79, was transferred into P. fluorescens strain Pf20118 by conjugation, as described in Materials and Methods, and the transconjugants were selected as blue colonies on PIA plates containing XGal and tetracycline. Double recombinants were selected as white and mucoid colonies on PIA plates containing XGal. These candidates were further tested for sensitivity to tetracycline. Twenty-four white, tetracycline-sensitive candidates were tested for PCR using the primer pair a/gF-1-fw and a/gF-2-Rev, as indicated in Table 2, and the products were analyzed by gel electrophoresis. PCR products from twenty-two of the candidates were the length expected for the wild-type a/gF allele (1.0 kb). However, the other two had the expected length for the mutant Aa/gF allele (0.7 kb). One of these was designated Pf20118a/gFA.

A deletion mutant of a/glJ was created by first creating a derivative (pMG49) of pMG27, from which a 1.4 kb A/rul-tfpa1 DNA fragment containing the 261 3' nucleotides of algl and the 5' 1140 nucleotides of a/gJ was removed. The deletion construct codes for an in-frame fusion of Alg\ and AlgJ which ensures that AlgF and AlgA should be translated normally. A 3.4 kb SacI-XbaI DNA fragment from pMG49 was then ligated into the suicide vector pHE55 digested with the same enzymes, creating pMG50. This plasmid, containing the sequences flanking the deletion, was introduced into Pf20118 by conjugation from E. coli S17.1 and non-mucoid transconjugants were selected on PIA medium with tetracycline. Transconjugant revertants were identified as mucoid tetracycline-sensitive colonies on LA medium. Four a/glJA mutant candidates were tested by PCR amplification of a region containing the deleted region using the primer pair Pface-tylFw and PfacetylRev (Table 2), and the PCR product was analyzed by agar-rose gel electrophoresis. Two of the colonies contained the wild-type fragment (1.8 kb), while the other two contained the mutant segment (0.4 kb). One of these was designated strain Pf20118a/glJA. Pf20118a/gFA and Pf20118a/glJA were cultured in fermentors using the PM5 medium and standard culture conditions, as set forth in Materials and Methods, and the alginate produced was harvested and measured as previously described. The results are given in table 4 below. Both varieties produced mannuronan alginate in yields of 16-17 g alginate per liter medium. The presence of acetyl groups was determined by<1>H-NMR spectroscopy, as described in Materials and Methods. Pf20118a/gFA did not produce acetylated alginate, while Pf20118a/glJA produced alginate containing small amounts of O-acetyl groups.

The fermentations were carried out in 3-liter fermenters using PM5 medium and standard cultivation conditions. Analyzes were performed as described in Material and Methods.

Pf20118a/gFA and Pf20118a/glJA have been deposited in NCIMB under accession numbers 41142 and 41143.

Example 5

Preparation of a modified derivative mutant strain showing low alginate lyase activity. Pf20118AlgLH203R.

P. fluorescens has, according to current knowledge, only one alginate lyase (AlgL) encoded by the gene a/gL. One option to control the molecular weight of the alginate produced by this bacterium is therefore to modify the AlgL gene product simultaneously produced.

The mutagenic primer pair algl_H203R1/algl_H203R2 was used to create a His203Arg-(H203R) mutation in the algL gene of Pf20118. The primers also contain passive mutations that create an Agel site, for allele identification. The mutagenic plasmid pMG70 was constructed, as described in Table 1, and introduced into the Pf20118 chromosome by conjugation, and transconjugants were selected on PIA medium with tetracycline and XGal. Transconjugants were grown as serial overnight cultures in the absence of tetracycline and plated on PIA medium with XGal to isolate AlgL mutants. White tetracycline-sensitive mutant candidates were screened by PCR amplification of the α/gL allele using the primers Pfa/gL-SspHI-pMG26 and Pfa/gl_Rev1 (Table 2), and alleles were identified by digesting The PCR fragment with Age\. The mutant strain selected was designated Pf20118>VgLH203R.

When the variant strain Pf20118Algl_H203R is grown in shake flasks using the reduced salt PIA medium, it yields amounts of mannuronan at approximately the same level as the variant strain Pf20118. Cultivation in a fermentor also led to approximately the same amounts of mannuronan produced from the two variant strains (H 2 O 3 R produced 12 g of mannuronan alginate per litre). The intrinsic viscosity measurements of the mannuronan produced by Pf20118 (intrinsic viscosity of 15 dl/g) and Pf20118Algl_H2O3R (intrinsic viscosity of 37 dl/g) in shake flasks (using reduced salt PIA medium, no proteases were added) show that the latter produces a mannuronan with increased molecular weight. Pf201 produced alginate with an intrinsic viscosity similar to Pf20118 (14 dl/g).

Bacterial cells of Pf201, Pf20118 and Pf20118Algl_H203R were harvested at the end of the fermentation in shaking flasks and ultrasonically treated. After ultrasound treatment, the extracts were examined for alginate lyase activity, which was measured by the method described in Materials and Methods. By defining the lyase activity of Pf201 as 100%, it is possible to detect activity down to 2% using this method. Pf20118 showed 93% activity. No activity was detected for Pf20118AlgLH203R, indicating that it is less than 2% of that of strain Pf201. Nevertheless, when the proteases Alkalase and Neutrase, both at 0.15 ml/l, were added, the intrinsic viscosity increased to approximately 50 dl/g for Pf201 and Pf20118, and to 70 dl/g for Pf20118AlgLH203R, indicating that the mutant lyase has some residual activity. The variant strain Pf20118AlgLH203R is deposited in NCIMB under accession number 41144.

Example 6

Production of variant mutant strains with reduced epimerase activity.

The mutant strains Pf201 and Pf20118 provide the means to produce alginate in vivo, with approximately 30% residual guluronate content, respectively pure mannuronan. However, alginates with intermediate amounts of guluronic acid (guluronate residue) content between 0 and 30% can also be formed. One way to obtain such strains is to delete the epimerase gene from the operon, and then introduce the gene controlled by a promoter either on a plasmid or a transposon. Plasmid pMG53 was constructed as described in Table 1, this plasmid contains a variant of algG in which the inner 40% of the gene has been removed. This plasmid was then transferred into Pf201, and a strain containing this deletion, designated Pf201 AalgG, was generated by homologous recombination. This strain did not form alginate, although it did form small oligouronides containing an unsaturated residue at the non-reducing end. Plasmid pCNB111 is a suicide plasmid, which contains a mini-transposon based on Tn5. Genes can be cloned into this mini-transposon in such a way that their expression is controlled by the inducible Pm promoter. The wild-type a/gG gene was transferred to this plasmid, as described in Table 1, creating plasmid pKB10. The plasmid was conjugated into Pf201Aa/gG, as described in the general description in Materials and Methods, under homologous recombination. But incorporation into the chromosome in this case depended on the transposon, not on homology. The resulting strain was designated Pf201Aa/gG::TnKB10. This strain produces alginate even in the absence of inducer, but the amount of polymer increases with increasing concentration of inducer (not shown). When the product was analyzed by NMR and mass spectrometry, it was found that the strain produces a mixture of alginate and oligomers. pKB10 was also transferred into Pf20118, creating strain Pf20118::TnKB10. This strain produces a mixture of wild-type alginate containing 30% G and mannuronan (results not shown). These results showed that not only is the epimerase required for alginate production, it is also epimerized as part of a protein complex. To obtain homogeneous alginate, only one form of epimerase can be present.

One method for producing variant strains with reduced epimerase activity is to replace wild-type algG with a mutant gene that codes for a mutant protein with reduced activity. A method has now been established to obtain such mutants, using the properties of Pf201Aa/gG and pKB10. Exponentially growing cells (OD6oonm:0.5) in E. coli S17.1 X-pir (pKB10) were mutagenized by nitrosoguanidine. Cells from 5 ml culture were washed twice in 5 ml TM buffer (1.0 g/l (NH4)2SO4, 0.1 g/l MgSO4x7H20, 5 mg/l Ca(NO3)2, 0.25 mg/ l FeS04x7H20, 5.8 g/l maleic acid, 6.05 g/l Tris, pH-adjusted to 6.0 with NaOH). The cells were resuspended in 2.85 ml TM buffer and treated with 100 µg/ml nitrosoguanidine at 37°C for 30 min. The suspension was cooled on ice for three minutes, the cells were pelleted by centrifugation and washed three times in 5 ml LB. Glycerol was added to a final concentration of 10% and the reslurry was frozen at -80°. Only 0.007% of the cells survived the mutagenesis. The plasmids were isolated from the thawed cells and transformed into E. coli S17.1 X-pir, in Table 1. The plasmids were now designated pKB10-M<*> to emphasize that they constitute a library of mutated versions of pKB10.

pKB10-M<*> was then conjugated to P. fluorescens Pf201Aa/gG as described in the Materials and Methods section (under homologous recombination), except that 100 fil frozen E. coli X-pir (pKB10-M<*> ) was inoculated directly into 10 ml of LB medium and grown for 2 h before being mixed with the P. fluorescens strain. The OD6oonm for both cultures was 0.4. After incubation on LA medium at 30°C for 36 hours, the conjugation mixture was plated on PIA medium (Difco) containing 40 µg/ml kanamycin (Km). Only those P. fluorescens where the transposon TnKB10-M<*> has been integrated into the chromosome will grow on this medium, because pKB10 and its derivatives

are unable to replicate in P. fluorescens. Even in the absence of inducers, some AlgG is expressed from the Pm promoter in P. fluorescens, and this level is sufficient to give mucoid colonies in strain Pf201Aa/gG::TnKB10. Approximately 0.5% of the colonies were non-mucoid, indicating either that the a/gG gene in these cells was not expressed due to mutations in either the Pm promoter or in the mRNA leader sequence, or that the AlgG protein is not - working.

Each plate (245 x 245 mm) contained 4-5000 colonies, and between 1200 and 1400 of these could be picked from each plate using an automated colony picker, Genetix Q-pixll, Genetix Limited, UK. The parameters were adjusted so that the small (non-mucoid) colonies were not picked. A method for screening the strain containing the mutated library was developed. In this screening, the parameters cell growth, alginate production (measured using a mixture of M and G lyases), respectively G content (measured using only G lyases), were measured;

Bacterial growth.

Colonies were replicated into two different liquid media in 96-well plates using a Genetix Q-pixll colony picker. To preserve the clones, one duplication was grown in 110 µl LB medium containing triclosan (0.025 g/l) and kanamycin (Km) (40 mg/l) and incubated for 48 hours at 25°C. Glycerol (60%, 40 µl) was added to each well, the solutions were mixed, and the plates were frozen at -SO₂C.

The other duplicates were grown in 0.5 X PIA (bacteriological peptone (10 g/l), NaCl (2 g/l), MgCl 2 (0.7 g/l), K 2 SO 4 (5 g/l), glycerol (5 g/l), triclosan (0.025 g/l) and Km (40 mg/l).) m-toluate (inducer) was added to 0.1 mM. The bacteria were grown in sterile Nunc 96-V well plates containing 110 µl medium/well, and incubated for 72 hours at 25°C using 900 rpm, orbital motion 3 mm amplitude.

Measurement of alginate production.

NaCl (0.2 M, 120 µl) was added to each well, and the cells were pelleted by centrifugation (3900 g, 20 µl, 30 min). 50 µl of supernatant from each well was transferred to a Nunc 96 flat-well plate. The alginate was deacetylated by adding NaOH (0.6M, 10 to each well, mixing for 25 seconds, and incubating at room temperature for 1 hour. 100 µl lyase reaction buffer (50 mM Tris-HCl, 1.5% NaCl ( pH 7.5)) was then added, and the solutions were mixed for 60 seconds. 75 µl from each well was transferred to a Costar-UV 96-well plate. An additional 150 µl of lyase reaction buffer was added, and the solutions were mixed in 25 seconds. The absorbance at 230 nm (A1) was read, then 8 µl of G-specific alginate lyase (0.2 u/ml) was added to each well. The solutions were mixed for 25 seconds and incubated at room temperature for 60 minutes. They were again mixed for 25 seconds and read at A230 nm (A2).Then 8 µM specific alginate lyase (1 µ/ml) was added to each well, the solutions were mixed for 25 seconds and incubated at room temperature for 60 minutes. The solutions were again mixed for 25 seconds and read at A230 nm (A3).The absorbance of the added lyases was subtracted from the readings A2 o g A3. Alginates of known composition (polymannuronic acid produced by Pf20118 and alginate with a G content of 30% produced by Pf201) were used as standards in the analyses.

The total amount of alginate is calculated by applying the formula:

Aaig<=>A3-A1

By comparing the Aaig from the samples with the Aaig from standards with known alginate concentration, the alginate concentration in the samples is calculated.

Measurement of guluronic acid (G) content.

The relative G content (Gr) in a sample is calculated using the formula

Gr= (A2-A1)/(A3-A1)

By comparing the Gr of the samples with the Gr of the standards, the G content of the samples was calculated.

Ten thousand colonies were screened in this way, and a few candidates with altered, but not zero, activity were picked. These mutants were then grown in shake flasks, as described under the Materials and Methods section. The PIA medium used was supplemented with triclosan (0.025 g/l), kanamycin (40 mg/l) and m-toluate (0.1 mM), and the alginate produced was analyzed using NM R, as described in materials and methods- the part. One mutant produced alginate containing only 13% guluronic acid residues, while the wild type produces alginate containing approximately 30% guluronic acid residues (Table 4). Some other strains that produce pure mannuronan were also found. The procedure makes it possible to screen mutant forms of AlgG that introduce less guluronic acid than the wild-type enzyme.

To produce additional variant mutant strains producing a desired alginate product, the mutant genes can be recovered using known PCR techniques, cloned into pMG48, and transferred into Pf201, or indeed any of the overproducing strains using homologous recombination, which previously described in the description. Similar to the mannuronan-producing strains: Pf2012, Pf2013, Pf20118 and Pf20137, a point mutation in algG that affects epimerization is unlikely to affect the amount of alginate produced.

Example 7

Production of variant mutant strains with reduced epimerase activity.

An alternative method for producing variant strains with reduced epimerase activity is to replace wild-type a/gG with a mutant gene that codes for a mutant protein with less activity. Four different amino acid substitutions are shown in Table 3 to give epimerase-negative mutants of AlgG. In these four cases, the amino acid change affects either the size of, or the charge of, the amino acid, for two of them both properties are changed. Possible additional amino acids can also be identified by sequencing mutants found by the method described in example 6. Alternative alleles of algG that code for more conservative changes in these amino acids are made by site-specific mutagenesis using pMG26 as a template. Mutagenic primers are made that contain a codon for the new amino acid flanked by approximately 10-15 nucleotides identical to the known sequence. Mutations in Ser337 will destroy the SmaI site, primers for the other amino acids preferably contain passive mutations that introduce a restriction enzyme site to help identify the new mutant strains. Primers for both strands must be synthesized, and the mutagenesis is performed as described in Material and Methods. Mutated α/gG alleles are then transferred into pMG48 digested with Nsi\-Nco\ as a 2.7 kb BspHI-PsfI digested DNA fragment. The resulting plasmids are transferred into Pf201 and transconjugants selected that are nonmucoid, tetracycline resistant, and blue on agar plates containing XGal. After culturing selected clones for several successive transfers in LB medium, double recombinants are selected that have white, mucoid colonies on agar plates containing XGal, and by being sensitive to tetracycline. algG from these candidates can be amplified using the primer pair Pfa/gG5f and Pfa/gG3r. The amplified product is 1.7 kb long. If a restriction site is removed, or introduced by the primers, the correct mutants are identified using the corresponding restriction enzyme. Alternatively, the candidates are confirmed by DNA sequencing.

The mutant strains are grown in shake flasks, and the alginate produced is isolated as described in Materials and Methods. The amount of alginate and the G content are determined using M and G lyases, as described in Materials and Methods. The results from interesting strains are confirmed by NMR spectroscopy.

Example 8

Preparation of variant mutant strain Pf201MC with an inducible Pm promoter for regulation of alginate production.

The Pm promoter, together with its effector protein XylS, is known to be a highly inducible promoter that can be used in many Gram-negative species, Blatny et al., 1997, 63, Appl. Environment. Microbiol., pp. 370-379. The inducer used is often toluate, which diffuses freely across the bacterial membranes. The P. fluore-scens strain Pf0-1 has now been sequenced at JGI (The DOE Joint Genome Insti-tute) ( http:// spider. jgipsf. org/ JGI microbial/ html/ Pseuctomonas/ pseudo homepa-ge. html) . When the alginate operon of this strain was compared with known alginate operon sequences from other Pseudomonas species, we found that the organization was similar. All sequenced alginate-producing species of Pseudomonas also have the same conserved open reading frame upstream of the alginate promoter. It potentially codes for a protein, the function of which is unknown. The purpose of this experiment was to replace the sequences downstream of the stop codon of this reading frame and upstream of the start codon of a/gD, the first gene of the alginate operon, with sequences encoding XylS, the Pm promoter and the Shine-Dalgarno sequence from the vector pJB658 described in Blatny et al., 1997, 38, pp. 35-51. Most of the DNA segment separating xylS and the Pm promoter in pJB658 was removed.

The first step was to clone the 3′ part of the hypothetical protein (abbreviated hyp) and the 5′ part of a/gD, to obtain flanking sequences for the insertion. When the sequences of a/gEGXLIJFA of strain PfO-1 were compared with those of NCIMB10525, it was found that the two sequences were not identical. The primers were therefore constructed using parts of the hyp and a/gD genes, which are well conserved in several species. The 3 part of hyp was cloned as a 0.7 kb BspLUI1 l-Spel digested PCR fragment using the primers HypBspLU111 and HypSpe\ in Table 2, into the suicide vector pMG48, generating pHE139. The 5′ portion of a/gD was cloned as a 0.8 kb A/ctel-A/s/l restricted PCR fragment into Nde\-Pst\-restricted pJB658celB, generating pHE138. The replacement vector pMC1, see Figure 5, was then constructed through a series of cloning steps (Table 1).

The plasmid was transferred by conjugation into strain Pf201, as described in Materials and Methods, selecting for XGal and tetracycline resistance/sensitivity to screen for recombinants and subsequent double recombinants. Colonies that appeared to be more mucoid on PIA medium containing 1 mM toluate than on PIA medium not containing toluate were selected for further analysis by PCR. Using the primer pair HypBspLU111 and AlgDNsi\ (Table 2), the expected PCR product from wild-type strains would be 2.3 kb long, while that of the mutant strain would be 3.0 kb. The selected mutant was designated Pf201 MC. This strain was then fermented in the absence and presence of toluate (0.025 mM) as an inducer. PM5 medium and standard conditions were used during the fermentation, as described in Material and Methods. The non-induced culture produced 3.5 g of alginate per liter of medium, while the induced culture produced 13 g of alginate per liter medium. The mutant strain Pf201 MC is deposited in NCIMB under the accession number 41145.

Example 9

Use of an inducible, mutated Pm promoter for regulation of alginate production.

The wild-type Pm promoter is indeed functional, but the non-induced level of expression is quite high. A method has been developed to screen for mutations in said promoter, based on the work of Winther-Larsen et al., Metabol. Meadow. (2000), 2, pp. 92-103. The original pJT19-bla was changed by inserting a termination sequence and an Afl\\\ site upstream of the Pm promoter and the Spel site downstream of the promoter was changed to a BspLU111 site. The new plasmid was designated p/B11. In this plasmid, the Pm promoter is flanked by unique Xba\ and BspLU111 restriction sites. Two complementary 50 bp DNA oligomers covering this DNA fragment were then synthesized. The conditions were chosen to give an error rate of approximately 12% in relation to the nucleotides flanked by these restriction sites. The corresponding wild-type strands were also synthesized. A library of double-stranded oligonucleotides was then made by hybridizing each of the oligonucleotides containing mutations with the complementary wild-type oligonucleotide. The ends of the oligonucleotides were designed to be complementary to p/B11 restricted by Xba\ and BspLU111. The hybridized oligonucleotides were then ligated to p/B11 restricted with Xba1 and BspLU111, and 50,000 transformants were obtained. In E. coli this library can be screened using resistance to ampicillin as a marker. But P. fluorescens already has a fairly high resistance to β-lactams. The gene for β-lactamase was replaced with a gene encoding luciferase, as described in Table 1, creating the vectors pHH100 containing the wild-type promoter and the pHH100 library containing the library of promoters. The pHH100 library contains 8000 independent transformants. It was then transferred to P. fluorescens by conjugation, as described in Materials and Methods.

The library of mutant Pm promoters was screened for luciferase activity using the assay described by Wood, K.V. and DeLuca, M. (1987, Anal.

Biochem. 161: 501-507). To obtain reproducible results, we found that the bacteria first had to be cultured in microtiter plates containing 110 µl of liquid PIA medium with 40 µg/ml kanamycin (Km) (25 µ, 48 hours, shaken at 90 rpm). Some bacteria were then diluted in fresh medium, using a sterile stamp ("stamp") for transfer, and then pressed onto two nylon filters. The filters were placed on PIA plates with or without inducer (1 mM m-toluate), the bacteria turned up, and incubated for 14 hours at SO2C. The filter was then placed in a Petri dish containing 3 ml of luciferin (Promega), (1 mM in 0.1 M sodium citrate, pH 5.0), shaken until the liquid was evenly distributed, and incubated for 10 minutes. It was placed on a filter paper to remove the liquid, and placed, face down, on transparent plastic film. A dry filter paper was placed on top of it to remove residual moisture. The nylon filter was then exposed for 10 minutes using a Kodak 2000IR camera. 1200 colonies were screened using this method, and 84 were identified that showed no or only weak activity from colonies grown without inducers, and readily detectable activity from colonies grown in the presence of inducers. Seventy-nine of these colonies were screened again, and seventy-five of them showed significantly lower activity in the absence of inducers, compared to wild-type pHHIOO.

Six of these clones were grown in 10 ml LB containing 50 mg/l kanamycin. Five were chosen because their expression levels without inducers were very low, the sixth (Pf201 (pHH100-E1)) had an intermediate expression level without inducers added, but also a significantly higher expression level in the presence of inducers. Stationary phase cultures (100 µl) were then transferred to two shake flasks containing 10 ml of fresh LB medium and incubated for two hours before addition of m-toluate to a final concentration of 1 mM. The cultures were harvested 14 hours after induction. Ninety jai culture was then added to 1.5 ml tubes containing 10 µl buffer (1M K 2 HPO 4 , 20 mM EDTA, pH 7.8) and frozen at -80°C. Luciferase activity was measured using the Luciferase Assay System from Promega Inc (Cat. No. E1500) (Table 5). The method proved useful in finding mutant promoters that not only achieved a very low non-induced expression level, but also had a low non-induced expression level and yet a fairly high induced expression level, compared to the wild type. The results are shown in the table below.

Strain NCIMB10525 was used as a blank and had no measurable activity. The average results from two independent inoculations of each strain are shown. a: Activity is given as arbitrary units (values depend on instrument settings), b: Activity in percent of percent of wild-type levels. c: Induced/non-induced values.

Example 10

In wfro epimerization of mannuronan alginate product.

Mannuronan, produced as described in Example 4, was dissolved in buffer (Mops (50 mM), CaCl2 (2.5 mM), NaCl (10 mM), pH 6.9) to a concentration of 0.25% mannuronan alginate . The mannuronan C5 epimerase AlgE4, produced and purified as described by Ramstad et al, Enzyme and Microbial Technology,

(1999), 24, pp. 636-646, was added to a concentration of 1 mg enzyme/200 mg mannuronan. The solution was incubated at 37°C for 23 hours. The epimerization was stopped by acid precipitation of the alginate. The alginate was then redissolved in distilled water and neutralized with alkali. To the alginate solution was added NaCl to a concentration of 0.2% and one volume of ethanol (96%) to precipitate the alginate. The precipitated alginate was washed 3 times with 70% ethanol, and 2 times with 96% ethanol, and freeze-dried. The freeze-dried alginate was further processed and analyzed using NM R, as described in Materials and Methods. The product after this incubation was an almost total poly-alternating alginate (PolyMG, Table 6).

Poly MG was redissolved in buffer (Mops (50 mM), CaCl 2 (2.5 mM), NaCl (10 mM), pH 6.9). The mannuronan C5 epimerase AlgE1 was produced and purified as described by Ramstad et al, Enzyme and Microbial Technology, (1999), 24, pp. 636-646, except that it was purified only by ion exchange chromatography. It was added to a concentration of 1 mg enzyme/200 mg mannuronan. The solution was incubated at 37°C for 4 days. Additional AlgE1 (1 mg enzyme/200 mg mannuronan) was added after 1, 2 and 3 days of incubation. The epimerization was stopped as described above. The alginate was isolated and analyzed by NMR, as described above. The result of the epimerization was an alginate with a G content of >95% (table 6).

Example 11

Alginate production using different carbon sources.

Pseudomonads are known to have the ability to utilize numerous compounds for growth. In this example, the ability to metabolize different carbon sources to alginate is demonstrated using the PM5 medium which replaces fructose with the relevant carbon source. The composition of the medium, the growth conditions and the analyzes of the alginate concentration in these fermentation experiments were carried out as described in the "general description of materials and methods", unless otherwise indicated.

The ability to produce alginate is shown for an alcohol (glycerol), monosaccharides (fructose, glucose) and for a disaccharide (lactose) (see Table 7).

P. fluorescens does not encode β-galactosidase, and is therefore unable to use lactose as a carbon source, although it can grow on both glucose and galactose. We transferred the plasmid pHM2, which encodes E. coli β-galactosidase (LacZ) and lactose permease (LacY), as described by Mostafa, H. E., Heller, K. J., Geis, A. 2002. Appl. Environment. Microbiol. 68: 2619-2623, to strain Pf201 by conjugation, as described in the homologous recombination chapter of the specification. The resulting strain is named Pf201(pHM2). To avoid problems with plasmid loss, lacZ and lacY could preferably be inserted into the chromosome using derivatives of plasmid pCNB111. Whey, a waste product from cheese production, contains large amounts of lactose. When Pf201 (pHM2) was grown in PM5 medium containing 27.5% ultrafiltered whey, corresponding to 50.9 g/l lactose in the medium, 13.3 g/l alginate was produced.

The results obtained are shown in table 7, and indicate that numerous carbon sources can be used to provide large amounts of alginate from the alginate-overproducing mutant strains.

<1>) The entire carbon source (40 g/l) is added before inoculating the fermentate.

<2>) 4.5 g/l glucose is added to the medium before the start of fermentation. The rest of the glucose (35.5 g/l) is fed at a continuous rate (1 g glucose/litre,

hour). The glucose feeding is started 10 hours after inoculation.

<3>) Ultrafiltered whey containing lactose as the main component and minerals as minor components is added to the PM-5 medium. The ultra-filtered whey also contains traces of milk proteins. The lactose concentration in the medium was 51 g/l after the addition of the ultrafiltered whey. The ultrafiltered whey was added before inoculating the fermentates.

Claims (33)

1. Mutant strain of P. fluorescens, characterized in that it is strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof, said strain producing at least 10 g of alginate per liter of medium and is stable over at least 60 generations. 2. Mutant strain of P. fluorescens according to claim 1, characterized in that said mutant strain produces at least 10 g of alginate per 40-55 g of carbon source per liter of medium. 3. Mutant strain of P. fluorescens according to claim 1, characterized in that said mutant strain produces at least 10 g of alginate per 50-55 g of carbon source per liter of medium. 4. Mutant strain of P. fluorescens according to claim 1, characterized in that said mutant strain produces at least 10 g of alginate per 40 g of carbon source per liter of medium. 5. Mutant strain of P. fluorescens according to claim 1, characterized in that said mutant strain is selected from the group consisting of mutant strain Pf201 deposited as NCIMB NO. 41137, Pf2012 deposited as NCIMB NO. 41138, Pf2013 deposited as NCIMB NO. 41139, Pf20118 deposited as NCIMB NO. 41140, Pf20137 deposited as NCIMB NO. 41141, Pf20118alglJA deposited as NCIMB NO. 41143, Pf20118algFA deposited as NCIMB NO. 41142, Pf20118Algl_H203R deposited as NCIMB NO: 41144 and Pf201MC deposited as NCIMB NO: 41145. 6. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that said mutant has the ability to produce an alginate consisting only of mannuronate residues only. 7. Mutant strain of P. fluorescens according to claim 5 or 6, characterized in that said mutant is selected from the group consisting of the mutant strains Pf2012 deposited as NCIMB 41138, Pf2013 deposited as NCIMB NO. 41139, Pf20118 deposited as NCIMB NO. 41140 and Pf20137 deposited as NCIMB 41141. 8. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that said mutant has the ability to produce alginate which has a defined guluronate residue (G) content of between 0 and 30%. 9. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that said mutant further comprises a mutant algF, algl and/or algJ gene which codes for an acetylase enzyme with reduced activity or which is inactivated and which thereby producing alginate without, or with a reduced number of O-acetyl groups. 10. Mutant strain of P. fluorescens according to claim 5 or 9, characterized in that said mutant is selected from the group consisting of the mutant strains Pf20118alglJA deposited as NCIMB NO. 41143 and Pf20118algFA deposited as NCIMB NO. 41142. 11. Mutant strain of P. fluorescens according to any one of claims 1 to 5, characterized in that said mutant has the ability to produce alginate with a molecular weight of between 50,000 and 3,000,000 Daltons. 12. Mutant strain of P. fluorescens according to claim 11, characterized in that said mutant is the mutant strain Pf20118Algl_H203R deposited as NCIMB 41144. 13. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that the mutant strain further comprises a mutant gene selected from the group consisting of: a mutant algG gene, a mutant algl gene, a mutant algJ gene, a mutant algL gene and a mutant algF gene. 14. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that the mutant strain further comprises a mutant algG gene which codes for an epimerase enzyme which has reduced epimerase activity. 15. Mutant strain of P. fluorescens according to any one of claims 1 to 4, characterized in that the mutant strain further comprises a mutant algG gene which is inactivated. 16. The mutant strain of P. fluorescens according to claim 1, characterized in that in said mutant strain, the alginate biosynthetic operon is regulated by an inducible promoter different from the naturally occurring promoter, and possibly one or more effector genes, in which said strain is obtained by a method comprising the following steps: (i) switching of the alginate biosynthetic operon promoter to a mutant strain of P.fluorescens, which is strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof, with an inducible promoter by homologous recombination, (ii) optionally introducing effector genes into the bacterium by (i) by homologous recombination, transposon mutagenesis or by means of a plasmid, (iii) culturing the bacterium produced in steps (i) and optionally step (ii) and then selection of mutants, (iv) selection from the mutants of (iii) a mutant that produces at least 10 g of alginate per liter of medium. 17. Mutant strain of P. fluorescens according to claim 16, characterized in that the inducible promoter is a Pm promoter or mutant thereof. 18. Mutant strain of P. fluorescens according to claims 16 or 17, characterized in that the inducible promoter is a Pm promoter, and further comprises the effector gene xylS. 19. Mutant strain of P. fluorescens according to any one of claims 16 to 18, characterized in that said mutant strain is Pf201 MC deposited as NCIMB NO. 41145. 20. Mutant strain of P. fluorescens according to any one of claims 16 to 19, characterized in that said mutant strain produces alginate as defined in any one of claims 6, 8, 9 and 11. 21. Mutant strain of P. fluorescens according to any one of claims 16 to 19, characterized in that said mutant strain further comprises a mutant gene as defined in any one of claims 13 to 15. 22. Mutant strain of P. fluorescens according to claim 20, characterized in that said mutant has the ability to produce an alginate consisting of only mannuronate residues. 23. Method for producing a mutant strain of P. fluorescens, characterized in that (a) a mutant strain of P. fluorescens, which is the strain Pf201 deposited as NCIMB NO. 41137, is contacted with a mutagenic agent, and (b) the treated bacterium in step (a) is grown in the presence of one or more antibiotics, and (c) antibiotic-resistant mucoid mutants are isolated by selection, and (d) the alginate production properties of the isolated the mucoid mutants in step (c) are determined. 24. Method according to claim 23, characterized in that the mutagenic agent is nitrosoguanidine. 25. Method according to claims 23 or 24, characterized in that the treated bacterium in step (a) is cultivated in the presence of a (3-lactam or aminoglycoside antibiotic. 26. Method according to claims 23 or 24, characterized in that the treated bacterium in step (a) is cultivated in the presence of carbenicillin. 27. Method for producing a mutant strain of P. fluorescens according to claim 16, characterized in that (i) the alginate biosynthetic operon promoter of mutant strain of P. fluorescens, which is strain Pf201 deposited as NCIMB NO. 41137 or a mutant thereof, is exchanged for an inducible promoter by homologous recombination, and (ii) is introduced into the bacterium from (i) by homologous recombination, transposon mutagenesis or by means of a plasmid, and (iii) mutants are grown and then isolated by selection, and (iv) the alginate production properties of the isolated mutants in (iii) are determined, and (v) a mutant producing at least 10 g of alginate per liter of medium is selected. 28. Method according to claim 27, characterized in that the inducible promoter is Pm from the P. putida Tol plasmid or a mutated Pm promoter. 29. Method for producing a mutant strain of P. fluorescens according to claim 8, characterized in that (a) the wild-type a/gG gene encoding the C-5 epimerase is cloned in a plasmid or minitransposon and mutagenized by chemical mutagenesis or by PCR to form a library of mutagenized algG genes, (b) a derivative of an alginate-producing strain of P. fluorescens lacking the a/gG gene (the Aa/gG strain) is constructed, (c) the library of mutagenized algG according to step (a) is transferred to the Aa/gG strain of P. fluorescens and the plasmid or transposon-containing strains are identified and analyzed for alginate production and epimerase activity, (d) plasmid or transposon-containing strains containing a mutant a /gG gene encoding an epimerase that provides alginate with a guluronic acid residue content between 0 and 30% is identified by the analysis in step (c), and (e) the mutant a/gG gene identified in step (d) is cloned into a gene replacement vector, and (f) gene-e The replacement vector of step (e) is then transferred to an alginate-producing strain of P. fluorescens to replace its a/gG gene with the mutant a/gG gene, enabling it to express the mutant gene. 30. Method for producing a mutant strain of P. fluorescens according to claim 8, characterized in that (a) one or more amino acids, which by mutagenesis and subsequent screening are identified as important for epimerization, are changed, at the gene level, by site-specific mutagenesis to amino acids different from those occurring in both the mutant and wild-type a/gG protein, and (b) the mutant gene is cloned into a gene replacement vector and this vector is transferred into an alginate-producing strain of P .fluorescence where it replaces the wild-type a/gG gene and is able to be expressed. 31. Use of at least one mutant strain of P. fluorescens according to any one of claims 1 to 22, for the production of alginate. 32. Use of at least one mutant strain of P. fluorescens according to any one of claims 1 to 22, for fermentor production of alginate on a large scale. 33. Use of the alginate consisting of only mannuronate residues produced by at least one mutant strain of P. fluorescens according to claim 6 or 22, in the manufacture of a food or industrial product including a pharmaceutical preparation, cosmetics, animal feed or food product, or as an intermediate for in vitro C-5 epimerization.