WO2015110218A1

WO2015110218A1 - Process for producing a beta-glucan polymer and genetically modified microorganisms useful in this process

Info

Publication number: WO2015110218A1
Application number: PCT/EP2014/077678
Authority: WO
Inventors: Andrea Hlubek; Herman Abel Bernhard WÖSTEN; Luis G. LUGONES; Jesse VAN VELUW
Original assignee: Wintershall Holding GmbH
Priority date: 2014-01-23
Filing date: 2014-12-15
Publication date: 2015-07-30
Also published as: RU2016134228A; KR20160108557A; ZA201605746B; BR112016016966A2; EP3097198A1; CA2937359A1; CN106414757A; MX2016009610A

Abstract

Process for producing a polymer consisting of a linear main chain of β-D-(1-3)-glucopyranosyl units having a single β-D-glucopyranosyl unit (1-6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, said process comprising the steps of: (i)culturing in a medium a genetically modified microorganism capable of producing a polymer consisting of a linear main chain of β-D-(1-3)-glucopyranosyl units having a single β-D-glucopyranosylunit (1-6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, wherein the modification confers adecreased activity - compared to a non-modified control microorganism of the same strain -of a hom2 gene product under conditions allowing said microorganism to produce said polymer; (ii)optionally recovering said polymer from the medium.

Description

PROCESS FOR PRODUCING A BETA-GLUCAN POLYMER AND GENETICALLY MODIFIED MICROORGANISMS USEFUL IN THIS PROCESS

The present invention relates to a process of producing beta-glucans herein also referred to as β-glucans, and to a process for producing beta glucan polymers by genetically modified microorganisms.

Technical Background

β-glucans are known well-conserved components of cell walls in several microorganisms, particularly in fungi and yeast.

A large number of closely related β-glucans exhibit a similar branching pattern such as schizophyllan, sderoglucan, pendulan, cinerian, laminarin, lentinan and pleuran, all of which exhibit a linear main chain of β-D-(1 -3)-glucopyranosyl units with a single β-D- glucopyranosyl unit (1 -6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3

WO 2014/006088 relates to a genetically modified microorganism capable of producing a polymer consisting of a linear main chain of β-D-(1 -3)-glucopyranosyl units having a single β-D-glucopyranosyl unit (1 -6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, characterized in that said genetically modified microorganism overexpresses (i) a polynucleotide encoding a polypeptide having 1 ,3^-D-glucan synthase-activity, and/or (ii) a polypeptide having 1 ,3^-D-glucan synthase-activity, compared to a corresponding non-modified control microorganism of the same strain

WO 2010/123350 relates to a fungus or a mushroom with an increased or decreased expression level of more than 200 polypeptides. In Example 4 a knock-out of the hom2 gene is described which leads to a phenotype of radial colony growth and no mushroom formation. Description of the Invention

In a first embodiment the invention relates to a process for producing a polymer consisting of a linear main chain of -D-(1 -3)-glucopyranosyl units having a single β-D- glucopyranosyl unit (1 -6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, said process comprising the steps of:

(i) culturing in a medium a genetically modified microorganism capable of producing a polymer consisting of a linear main chain of β-ϋ-(1 -3)- glucopyranosyl units having a single β-D-glucopyranosyl unit (1 -6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, wherein the modification confers a decreased activity - compared to a non-modified control microorganism of the same strain - of a hom2 gene product

under conditions allowing said microorganism to produce said polymer; (ii) optionally recovering said polymer from the medium.

The hom2 gene product is a homeodomain protein encoded by the hom2 gene. A preferred polypeptide sequence for the hom2 gene product is described in Ohm et al. 2010, Genome sequence of the model mushroom Schizophyllum commune, Nature Biotechnology 28: 957-963, (protein ID 257987) which is disclosed in SEQ ID NO:3. Another preferred hom2 gene product is disclosed in SEQID NO:4.

BLAST search results comprise homeodomain proteins in general. Examples with homologous sequences in addition to the homeobox can be found in Coprinopsis cinerea, Laccaria bicolor, Postia placenta, Serpula lacrymans, Phanerochaete carnosa, Agaricus bisporus.

A genetically modified microorganism having a modification which confers a decreased activity of a hom2 gene product shall mean a microorganism which is treated by endogenous or exogenous factors in order to decrease at the end the activity of the corresponding gene product.

This is possible at different levels, e.g. at the level of the DNA, at the level of the RNA and at the level of the protein. At the level of the DNA a decreased activity of the respective gene product can be achieved by deleting partly or completely the hom2 gene in the genome of the microorganism. A complete deletion - also known as knock-out - of hom2 can lead to a complete extinction of the hom2 gene product in the microorganism if no other alleles or copies of the hom2 gene are present. In dikaryons a complete deletion means a double knock-out of the hom2 gene in both nuclei. A partial deletion of the hom2 gene can be achieved by deleting only fragments (parts) of the hom2 gene or by deleting only one allele or copy in a genome carrying further hom2 genes. If only fragments of the hom2 gene are deleted, it is possible to maintain a residual activity of the hom2 gene product, depending on the position and / or the length of the deleted fragments. However, also by partial deletion a complete extinction of the hom2 gene product activity is possible, e.g. by deleting a single base-pair resulting in a frameshift during the translation. At the level of RNA it is also possible to decrease the gene expression by RNA interference (RNAi), by which gene expression of the hom2 gene can be inhibited. This can be achieved by constructing micro RNA (miRNA) or small interfering RNA (siRNA) which bind to mRNA transcribed from hom2 gene and inhibits or destroys this mRNA. At the level of RNA it is also possible to decrease the gene expression by using genetic elements such as weak or transient instead of strong or permanent promoters, enhancers, terminators and other regulatory elements which allow a lower or only a transient gene expression of the respective gene. Another possibility is to destabilize the RNA transcripts made of the respective gene in order to effect a lower number of transcripts available for translation per time unit.

Another possibility to interfere with at the protein side is the codon usage of the gene. If a codon usage with rare codons in the respective microorganism is used, a lower translation rate resulting in a decrease of active protein is effected compared to a non modified microorganism.

In the context of this invention the term "genetically modified microorganism" should be understood in a broad sense; not only "genes" and "genetic elements" such as promotors, are encompassed by the term "genetically modified microorganism" , also a repression of gene regulation by using molecules (repressors) binding tightly to an operator and thus inactivating the gene expression is understood as a genetically modified microorganism according to this invention.

The decrease in activity of the hom2 gene product is compared to a non-modified microorganism. This shall mean that both microorganisms - the modified and the non- modified - shall have the same genetic background and shall be treated under the same physiological conditions with the exception of that measure which effects the decrease of activity of the hom2 gene product. For example, in case of a knock-out of hom2 gene in S. commune, the non-modified reference organism shall be a S. commune organism having the same genetic background except of the hom2 gene knock-out and which is treated under the same conditions as the knock-out organism . Non-limiting examples of suitable microorganisms useful as starting organisms for the genetic modification according to the invention are microorgansims of the genus Schizophyllum especially Schizophyllum commune, Sclerotium especially, Sclerotium rolfsii, Sclerotium glucanicum, Sclerotium delphinii, Porodisculus especially Porodisculus pendulus, Botrytis especially Botrytis cinerea, Laminaria especially Laminaria sp., Lentinula especially Lentinula edoles, and Monilinia especially Monilinia fructigena.

Preferred microorganisms for the process according to the invention are Schizophyllum commune which are availabe from public deposits, e.g.:

DSM-1024, DSM-1025, DSM-1026, DSM-1 1223

ATCC® Number: 204191 , ATCC® Number: MYA-2104, ATCC® Number: 26890, ATCC® Number: 26892, ATCC® Number: 62873, ATCC® Number: 38229,

ATCC® Number: 32746, ATCC® Number: MYA-4819, ATCC® Number: 52396,

ATCC® Number: MYA-1 128, ATCC® Number: 42093, ATCC® Number: 18246,

ATCC® Number: MYA-1 124, ATCC® Number: 38230, ATCC® Number: 26889,

ATCC® Number: 26262, ATCC® Number: 52398, ATCC® Number: MYA-1 123. In another embodiment the invention relates to a process for producing a polymer as disclosed above by culturing any of the genetically modified microorganisms disclosed above. The genetically modified microorganisms according to the invention can be produced by known techniques of recombinant DNA technology such as described e.g. in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994-1998) ). Further examples for the construction of genetically modified microorganisms are disclosed in the Experimental Part.

Methods for culturing microorganisms such as fermentation processes are known in the art and also described and exemplified herein (Kumari, Bioresource Technol (2008), 99: 1036-1043; Reyes, J Natural Studies (2009), 7(2), January-June). In context with the present invention, such methods allow the respective microorganism to grow and to produce the desired β-glucan as described and exemplified herein. Suitable media may comprise, e.g., coconut water as described in Reyes, loc cit. Furthermore, as known in the art, there are several media particularly suitable for particular microorganisms.

For example, also in context with the present invention, suitable media for culturing S. commune comprise CYM medium (25 g agar (Difco), 20 g glucose (Sigma), 2 g trypticase peptone (Roth), 2 g yeast extract (Difco), 0.5 g MgSO₄ x 7 H₂O (Roth), 0.5 g KH₂PO₄ and 1 g K₂HPO₄ (both from Riedel-de Haen) per liter H₂O) (particularly useful for cultivation on solid support) or a medium comprising 30 g glucose (Sigma), 3 g yeast extract (Difco), 1 g KH₂PO₄ (Riedel-de Haen), 0.5 g MgSO₄ x 7 H₂O (Roth) per liter H₂O (particularly useful for liquid cultures) as also described and exemplified herein.

In context with the present invention, the term "average branching degree about 0,3" may mean that in average about 3 of 10 -D-(1 -3)-glucopyranosyl units are (1 -6) linked to a single β-D-glucopyranosyl unit. In this context, the term "about" may mean that the average branching degree may be within the range from 0.1 to 0.5, preferably from 0.2 to 0.4, more preferably from 0.25 to 0.35, more preferably from 0.25 to 0.33, more preferably from 0.27 to 0.33, and most preferably from 0.3 to 0.33. It may also be 0.3 or 0.33. Schizophyllan, scleroglucan, pendulan, cinerian, laminarin, lentinan and pleuran all have an average branching degree between 0.25 and 0.33; for example, scleroglucan and schizophyllan have an average branching degree of 0.3 to 0.33 (Survase, loc cit; Novak, loc cit). The average branching degree of a β-glucan can be determined by methods known in the art, e.g., by periodic oxidation analysis, methylated sugar analysis and NMR (Brigand, Industrial Gums, Academic Press, New York/USA (1993), 461 -472).

In one embodiment of the present invention, the polymer to be produced is selected from the group consisting of schizophyllan, scleroglucan, pendulan, cinerian, laminarin, lentinan and pleuran. For example, the polymer may be schizophyllan or scleroglucan, particularly schizophyllan.

The recovering of the polymer from the fermentation product can be performed by a number of routine techniques known in biotechnology such as precipitation and centrifugation.

Many processes for the preparation of β-glucans comprise the cultivation and fermentation of microorganisms capable of synthesizing such biopolymers. For example, EP 271 907 A2, EP 504 673 A1 and DE 40 12 238 A1 disclose processes for the preparation, i.e. the preparation is effected by batchwise fermentation of the fungus Schizophyllum commune with stirring and aeration. The culture medium substantially comprises glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water. EP 271 907 A2 describes a method for isolating the polysaccharide, in which the culture suspension is first centrifuged and the polysaccharide is precipitated from the supernatant with isopropanol. A second method comprises a pressure filtration followed by an ultrafiltration of the solution obtained, without details of the method having been disclosed. "Udo Rau, "Biosynthese, Produktion und Eigenschaften von extrazellularen Pilz-Glucanen", Habilitationsschrift, Technical University of Brunswick, 1997, pages 70 to 95" and "Udo Rau, Biopolymers, Editor A. Steinbuchel, Volume 6, pages 63 to 79, WILEY-VCH Publishers, New York, 2002" describe the preparation of schizophyllan by continuous or batchwise fermentation. "GIT Fachzeitung Labor 12/92, pages 1233 - 1238" describes a continuous preparation of branched β-1 ,3-glucans with cell recycling. WO 03/016545 A2 discloses a continuous process for the preparation of scleroglucans using Sclerotium rolfsii. Furthermore, for economic reasons, the concentration of aqueous β-glucan solutions should be as high as possible in order to ensure as little transport effort as possible for transporting the aqueous glucan solutions from the production site to the place of use. For this purpose, β-glucan solutions are usually concentrated by drying, lyophilization and/or precipitation before being transported in order to reduce their weight.

The genetically modified microorganism according to the invention is able to produce at least 1 .5 times, more preferably at least 1 .8 times more, more preferably at least 2.0 times more, and most preferably at least 2.2 times more β-glucan polymer compared to the corresponding non-modified control microorganism. In this context, production of, e.g., 1 .5 times "more" β-glucan polymer may mean that a genetically modified microorganism produces an amount of β-glucan polymer which is 1 .5 times higher compared to the amount of β-glucan polymer produced in the same time under the same conditions by a corresponding non-modified control microorganism. Alternatively, production of, e.g., 1 .5 times "more" β-glucan polymer may mean that a genetically modified microorganism produces the same amount of β-glucan polymer as a corresponding non-modified control organism under the same conditions, however, 1 .5 times faster. The amount of produced β-glucan polymer may be measured by methods known in the art and as also described herein.

Experimental Part

Table 1. Strains used

Dikaryotic Strains Parental background Reference

H₄-8 x H4-8b - Ohm et al. 201 Oab

Ahom2 Ahom2 H₄-8 x H4-8b Ohm et al. 201 1

Ahom2 Ahom2::hom2 (c2) Ahom2 Ahom2 This study; for experimental details see

Ohm et al. 201 1

Ahom2 Ahom2::hom2 (c7) Ahom2 Ahom2 This study; for experimental details see

Ohm et al. 201 1

Afst4 Afst4 H₄-8 x H4-8b Ohm et al. 2010

AWC2 AWC2 H₄-8 x H4-8b Ohm et al. 2013

Strains and culture conditions for Example 1

Dikaryotic S. commune strains (Table 1 ) were grown for 7 days in the dark at 25 °C on minimal medium (MM) agar (1 .5%) plates from a point inoculum (Dons JJM et al. (1979). Characterization of the genome of the basidiomycete Schizophyllum commune. Biochimica et Biophysica Acta 563: 100-1 12.). A quarter of the colony was homogenized in a Waring blender at full speed for 30 seconds in 50 ml liquid MM. The 50 ml homogenate was incubated at 25 °C and 200 rpm in a 250 ml Erlenmeyer flask in the dark. After 24 h, the pre-culture was homogenized (see above) and 2 ml was centrifuged. The supernatant was discarded and the wet weight of the pellet was determined. From this, the volume of the homogenate was calculated that contains 0.2 g wet weight mycelium. This volume was used to inoculate 100 ml liquid shaken cultures using MM and 250 ml Erlenmeyer flasks. Cultures were grown in the dark at 25 °C and 200 rpm in triplicate or even more replicates.

Strains and culture conditions for Example 2

Colonies of the dikaryotic strains H4-8 and Ahom2Ahom2 were grown from a point inoculum for 7 days in the dark on MM agar plates. Pieces of 2 χ 3 cm² were excised from the intermediate zone of these colonies. Agar was removed as much as possible and the mycelium was homogenized in 15 ml DHSV medium (33 g ¹ glucose monohydrate, 0.5 g ¹ MgSO₄-7H₂O, 0.9 g ¹ KH₂PO₄, 0.1 g ¹ K₂HPO₄, 0.5 g ¹ citric acid, 50 g ¹ urea, pH 5.8. This is supplemented by spore elements: 20 mg ¹ FeSO₄-7H₂O, 50 mg ¹ Titriplex III (Merck Millipore), 1 mg ¹ ZnSO₄-7H₂O, 0.3 mg ¹ MnSO₄-4H₂O, 3 mg ¹ H₃BO₃, 2 mg ¹ CoCI₂-6H₂O, 0.1 mg ¹ CuCI₂-2H₂O, 0.2 mg ¹ NiCI₂-6H₂O, 0.3 mg ¹ Na₂MoO₄-2H₂O and vitamins: 0.0105 mg ¹ Biotin, 0.0045 mg ¹ Folic acid, 2.31 mg ¹ 4-Aminobenzoic acid, 0.18 mg ¹ Riboflavin, 0.825 mg ¹ Calcium pantothenate, 1 .8 mg ¹ Nicotinamide, 0.135 mg ¹ Pyridoxine-HCI, 4.2 mg ¹ Myoinositol, 1 .605 mg ¹ Thiamine-HCI) for 1 minute at 6,000 rpm using a T 25 digital ULTRA-TURRAX (IKA). 2 50 ml pre-cultures per strain were inoculated with 2.5 ml homogenate. To this end, 50 ml DHSV medium had been added in 100 ml Erlenmeyers. Cultures were grown at 30 °C and 180 rpm for 72 h in the dark. The pre-cultures were centrifuged for 10 min at 9,935 g and the pellets of each strain were pooled. The supernatant was collected and glucose and glucan was measured as described below. The pooled pellets were homogenized again in 25 ml DHSV (see above). 50 ml DHSV cultures were inoculated with 2.5 ml homogenate using 100 ml Erlenmeyers. Cultures were grown at 30 °C and 180 rpm for 3, 8 or 10 days in triplicate in the dark.

Viscosity of the medium for Example 1 Culture medium was collected by centrifugation at 5,000 g for 10 minutes or filtration over Miracloth. Formic acid (4 g ¹ final concentration) was added to stabilize the medium and the viscosity was measured. To this end, a 10 ml BD Plastipak syringe with a Greiner Bio One blue pipet tip (1000 μΙ) attached to its nozzle was filled with culture medium. The time it took for 8 ml of the fluid to flow out of the syringe (upper level of the medium from 10 ml marker position to 2 ml marker position) was used as a measure of viscosity. This procedure was related to measurements with a HAAKE Rheostress 1 Rotational Rheometer 1 (Thermo Fischer). Measurements correlated with a Pearson's correlation coefficient of more than 0.95.

Glucan, urea, ethanol and biomass measurements for Example 1 and / or 2

To determine SPG production, 10 ml of culture was pipetted in a 50 ml Falcon tube and stabilized with 3 g ¹ ACTICIDE BW20 (Thor) and treated with 10 ml ¹ Submers β- Glucanase from Penicillium funiculosum NS (Erbsloh) for 24 h at 40 °C. After centrifugation at 3,400 g for 30 minutes the ethanol and glucose concentration in the supernatant was measured using a HPLC cation exchanger (Aminex HPX-87 H, Bio- Rad) with 5 mM H₂SO₄ (Roth) as eluent and 0.5 ml min^"1 flow rate at 30 °C. The concentration of urea in the culture medium was also measured by HPLC. To this end, a fast carbohydrate column, 100 7.8 mm (Biorad) was used with water as eluent and a 1 ml min^"1 flow rate at 70 °C.

To determine the biomass produced in the cultures the pellet obtained after centrifugation at 3,400 g for 30 minutes (see above) was resuspended in 30 ml H₂O and shaken vigorously. After centrifugation at 3,400 g for 30 minutes the pellet was again suspended in 30 ml H₂O by shaking and placed on Whatman filter paper, rinsed twice with 50 ml water and then vacuum dried. A Mettler Toledo HB43-S Halogen Moisture Analyzer was used to further dry and weigh the samples. The weight of the filter (determined before the mycelium was placed on the filter) was subtracted.

Example 1

Viscosity of liquid shaken cultures of the wild-type H4-8 H4-8b dikaryon and its derivative the Ahom2Ahom2 strain was measured during a 10-days cultivation period. Viscosity of the medium was higher in the Ahom2Ahom2 strain when compared to the wild-type from day 5 and day 6 onwards as shown by the reduced flow rate of the culture medium out of a syringe (Figure 1 ) and by a Rotational Rheometer 1 (Figure 2), respectively. Viscosity of the medium was related to SPG production by glucan analysis. To this end, the culture was subjected to degradation by glucanase and the resulting glucose was quantified by HPLC analysis (Figure 3). Glucose release by glucanase activity was detected after 8 days and 10 days of culturing of the Ahom2Ahom2 strain and the wild-type strain, respectively. At day 10 glucose levels were 5 times higher in the Ahom2Ahom2 strain. Taken together, the Ahom2Ahom2 strain produces more SPG as indicated by viscosity and glucanase experiments.

The viscosity of the medium was also measured for the AWC2AWC2 and Afst4Afst4 strains and two complemented Ahom2Ahom2 strains (Figure 4, 5). Viscosity of the medium for all four strains was not significantly different from that of the wild-type. Taken together, these data confirm that absence of Hom2 increases schizophyllan production. In contrast, absence of Fst4 and WC2 does not affect SPG production despite the fact that these strains are unable to form fruiting bodies like the Ahom2Ahom2 strain. Example 2

Viscosity, glucan, glucose (added as carbon source), urea, ethanol and biomass were quantified in 3-, 8-, and 10-day-old cultures of the Ahom2Ahom2 and wild type S. commune dikaryons (Figure 6, 7 & 8). The culture medium of the Ahom2Ahom2 strain was more viscous than that of the wild-type at day 8 and 10 as measured by a rheometer (Figure 6). The amount of glucose released by glucanase was significantly higher after 10 days of culturing in the Ahom2Ahom2 strain when compared to the wild- type strain (Figure 7). The Ahom2Ahom2 strain showed less ethanol production at day 8 (two-fold difference with wild-type) and this metabolite was even absent after 10 days (Figure 8). In contrast, the wild type had more than doubled the amount of ethanol from day 8 to 10 (Figure 8). At day 10, glucose and urea were totally consumed in the case of the Ahom2Ahom2 dikaryon but this was not the case for the wild type (Figure 8). Yield of SPG per biomass was 20 % higher in the Ahom2Ahom2 strain when compared to the wild-type (Figure 9). Moreover, the Ahom2Ahom2 strain produced 3.7 times as much SPG per glucose than the wild-type. We conclude that the S. commune Ahom2 dikaryon is superior in producing SPG when compared to the parent.

List of figures :

Figure 1. Viscosity of the culture medium of liquid shaken cultures of wild type and Ahom2Ahom2 dikaryons of S. commune as measured by the flow rate through a syringe.

Figure 2. Viscosity (mPa-s) of the culture medium of liquid shaken cultures of wild type and Ahom2Ahom2 dikaryons of S. commune. Viscosity was measured with a rheometer using the same samples as in Figure 1 . Figure 3. Glucan analysis of the culture medium from liquid cultures of wild type and Ahom2Ahom2 dikaryons of S. commune. Glucan was quantified by measuring glucose release resulting from glucanase treatment. Samples are identical to those of Figure 1 and 2. Figure 4. SPG production per gram biomass in 10-day-old liquid cultures of the dikaryotic wild type strain H4-8 H4-8b and the dikaryotic knockout strains Ahom2Ahom2, AWC2AWC2, and Afst4Afst4.

Figure 5. SPG production per gram biomass in 10-day-old liquid cultures of the dikaryotic wild type strain H4-8 H4-8b, the dikaryotic knockout strain Ahom2Ahom2, and two complemented Ahom2Ahom2 strains in which hom2 was reintroduced in one of the parental nuclei.

Figure 6. Viscosity (mPa-s) of culture medium of liquid shaken cultures of wild type and Ahom2Ahom2 dikaryons of S. commune. Viscosity was measured with a rheometer. Figure 7. Glucan analysis of culture medium from liquid cultures of wild type and Ahom2Ahom2 dikaryons of S. commune. Glucan was quantified by measuring glucose release resulting from glucanase treatment. Samples are taken from the same cultures as those of Figure 6.

Figure 8. Glucose, urea and ethanol levels in culture medium of liquid shaken cultures of wild type and Ahom2Ahom2 dikaryons of S. commune. Samples are identical to those of Figure 6. Figure 9. SPG yield per biomass (gram / gram) and per glucose (gram / gram) of the dikaryotic wild-type strain of S. commune and its derivative the Ahom2Ahom2 strain.

SEQ ID NO:1 is a polynucleotide sequence of hom2 gene from Schizophyllum commune used in the experimental part. SEQ ID NO:2 is a polynucleotide sequence of hom2 gene from Schizophyllum commune which is slightly different from the one disclosed in SEQ ID NO:1 .

SEQ ID NO:3 is the polypeptide sequence of hom2 gene from Schizophyllum commune translated from SEQ ID NO:1

SEQ ID NO:4 is the polypeptide sequence of hom2 gene from Schizophyllum commune translated from SEQ ID NO:2 References

Dons JJM, de Vries OMH, Wessels JGH (1979). Characterization of the genome of the basidiomycete Schizophyllum commune. Biochimica et Biophysica Acta 563: 100-1 12.

Niederpreum DJ, Marshall C, Speth JL (1977). Control of extracellular slime accumulation in monokaryons and resultant dikaryons of Schizophyllum commune. Sabouraudia 15: 283-295.

Ohm RA, de Jong JF, Lugones LG, Aerts A, Kothe E, Stajich JE, de Vries RP, Record E, Levasseur A, Baker SE, Bartholomew KA, Coutinho PM, Erdmann S, Fowler TJ, Gathman AC, Lombard V, Henrissat B, Knabe N, Kues U, Lilly WW, Lindquist E, Lucas S, Magnuson JK, Piumi F, Raudaskoski M, Salamov A, Schmutz J, Schwarze FWMR, vanKuyk PA, Horton JS, Grigoriev IV, Wosten HAB (2010a). Genome sequence of the model mushroom Schizophyllum commune. Nature Biotechnology 28: 957-963.

Ohm RA, de Jong JF, Berends E, Wang F, Wosten HAB, Lugones LG (2010b). An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune. World Journal of Microbiology & Biotechnology 10: 1919- 1923.

Ohm RA, de Jong JF, de Bekker C, Wosten HAB, Lugones LG (201 1 ). Transcription factor genes of Schizophyllum commune involved in regulation of mushroom formation. Molecular Microbiology 81 : 1433-1445. Ohm RA, Aerts D, Wosten HA, Lugones LG (2013). The blue light receptor complex WC-1/2 of Schizophyllum commune is involved in mushroom formation and protection against phototoxicity. Environmental Microbiology 15: 943-955.

Claims

Process for producing a polymer consisting of a linear main chain of β-ϋ-(1 -3)- glucopyranosyl units having a single β-D-glucopyranosyl unit (1 -6) linked to a β- D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, said process comprising the steps of:

under conditions allowing said microorganism to produce said polymer;

(ii) optionally recovering said polymer from the medium.

Process according to claim 1 , wherein said polymer is selected from the group consisting of schizophyllan, sderoglucan, pendulan, cinerian, laminarin, lentinan and pleuran.

Process according to claiml , wherein the decreased activity of the hom2 gene product is effected by a gene knock-out of the hom2 gene.

Process according to claim3, wherein the genetically modified microorganism is a dikaryon of Schizozophyllum.

Process according to claim4, wherein the dikaryon carries a double knock-out of hom2 gene.