KR20160108557A - Process for producing a beta-glucan polymer and genetically modified microorganisms useful in this process - Google Patents

Abstract

(1-3) -glycopyranosyl unit having a single? -D-glucopyranosyl unit linked to a linear main chain? -D-glucopyranosyl unit having an average molecular weight of about 0.3 (1-6) Of a linear backbone comprising the steps of:
(i) β-D- (1-3) -glucose having a single β-D-glucopyranosyl unit linked to a linear main chain β-D-glucopyranosyl unit having an average branching of about 0.3 (1-6) A genetically engineered microorganism capable of producing a polymer consisting of a linear main chain of a pyranosyl unit, wherein said microorganism is a microorganism that confers a reduced activity of the hom2 gene product compared to an untreated control microorganism of the same strain, Culturing under conditions permitting production of 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 < RTI ID = 0.0 >

The present invention relates to a method for producing beta-glucan, also referred to herein as beta -glucan, and a method for producing beta-glucan polymer by genetically engineered microorganisms.

β-Glucan is a well-known and well-preserved component of the cell wall in many microorganisms, particularly in fungi and yeast.

Many closely related [beta] -glucans exhibit similar branching patterns, such as, for example, zymo-pilan, scleroglucan, penluan, cynerian, laminarin, lentanan and furan, (1-3) -glycopyranosyl unit having a single? -D-glucopyranosyl unit linked to the? -D-glucopyranosyl unit (1-6).

WO 2014/006088 discloses β-D- (1-3) β-D-glucopyranosyl units having a single β-D-glucopyranosyl unit linked to a linear backbone β-D-glucopyranosyl unit having an average branching index of about 0.3 (1-6) - (i) a polypeptide having 1,3 -? - D-glucan synthase activity, and / or (ii) a polypeptide having 1,3-ss-glucan synthase activity characterized in that the polynucleotide encoding a polypeptide having -β-D-glucan synthase activity is over-expressed relative to the corresponding untreated control microorganism of the same strain.

WO 2010/123350 relates to increased or decreased levels of expression of more than 200 polypeptides or fungi. It is described in Example 4 that the knock-out of the hom2 gene results in a phenotype of radial colony growth and mushroom non-formation.

In a first embodiment, the present invention relates to β-D- (1- (2-hydroxyethyl) -1,2,3,4-tetrahydroisoquinolin-2-yl) 3) -glucopyranosyl unit, comprising the steps of:

(i) (1-3) -glycopyranosyl unit having a single? -D-glucopyranosyl unit linked to a linear main chain? -D-glucopyranosyl unit having an average molecular weight of about 0.3 (1-6) Wherein the microorganism is a microorganism that confers reduced activity of the hom2 gene product compared to an untreated control microorganism of the same strain, wherein the microorganism is capable of producing the polymer Culturing under conditions permitting production;

(ii) Optionally recovering the polymer from the medium.

The hom2 gene product is a homeodomain protein encoded by the hom2 gene. Preferred polypeptide sequences for the hom2 gene product are 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 SEQ ID NO: 4.

BLAST search results generally include homeodomain proteins. Examples of homologous sequences in addition to homeobox include Coprinopsis cinerea, Laccaria bicolor, Postia placenta, Serpula lacrymans, It can be found in Phanerochaete carnosa, Agaricus bisporus.

A genetically engineered microorganism having an operation that confer reduced activity of the hom2 gene product will eventually mean a microorganism that is treated by an endogenous or exogenous factor to reduce the activity of the corresponding gene product.

This is possible at different levels, for example at the level of DNA, at the level of RNA and at the level of protein.

The reduced activity of each gene product at the level of DNA can be achieved by partially or completely eliminating the hom2 gene in the genome of the microorganism. Complete deletion of hom2 - also known as rust-out - can result in complete extinction of the hom2 gene product in the microorganism if no other allele or copy of the hom2 gene is present. Complete deletion in the nucleus means double knockout of the hom2 gene in both nuclei. A partial deletion of the hom2 gene can be accomplished by deleting only one segment (portion) of the hom2 gene or by deleting only one allele or copy in the genome harboring an additional hom2 gene. If only the segment of the hom2 gene is deleted, it is possible to maintain the residual activity of the hom2 gene product depending on the position and / or length of the deleted segment. However, complete deletion of the hom2 gene product activity by partial deletion is also possible, for example by deleting a single base pair, resulting in a frame shift during translation.

It is also possible to reduce gene expression by RNA interference (RNAi) at the level of the RNA (whereby gene expression of the hom2 gene may be inhibited). This can be achieved by constructing microRNAs (miRNAs) or small interfering RNAs (siRNAs) that bind to mRNA transcribed from the hom2 gene and inhibit or destroy such mRNA.

Reducing gene expression by using weak or transient promoters, enhancers, terminators, and other regulatory elements that allow lower or only transient gene expression of each gene, instead of genetic elements such as strong or permanent at the level of RNA, It is possible.

Another possibility is to destabilize RNA transcripts made with each gene to produce a lower number of transcripts available for translation per unit of time.

Another possibility of interference on the protein side is the codon usage of the gene. When codon usage preferences for rare codons are used in each microorganism, a lower translation rate results, resulting in a decrease in active protein relative to untreated microorganisms.

The term "genetically engineered microorganism" in the context of the present invention will be understood in a broad sense; Not only are "genes" and "genetic elements" such as promoters encompassed by the term "genetically engineered microorganisms", but also by the use of molecules (inhibitors) that tightly bind to an operative gene and inactivate gene expression Inhibition is understood as a genetically engineered microorganism according to the present invention.

A decrease in the activity of the hom2 gene product is compared to untreated microorganisms. This would mean that both microorganisms-engineered and untreated-would have the same genetic background and would be processed under the same physiological conditions, except for measures that would result in a decrease in the activity of the hom2 gene product. For example, in the case of the knock-out of the hom2 gene in the Sijo philomicum, the untreated reference organism has the same genetic background, except for the green-out organism and the hom2 gene knock-out, It would be a Philomack organism.

Non-limiting examples of suitable microorganisms useful as starting organisms for genetic manipulation according to the present invention include microorganisms of the genus Schizophyllum , in particular Schizophyllum commune , Sclerotium , (Sclerotium rolfsii), Scotland Klee Loti help glue Carney Qom (Sclerotium glucanicum), Scotland Klee Loti help Delphi kneader (Sclerotium delphinii), prisoners di School of Ruth (Porodisculus), especially prisoners di school Ruth pendul Ruth (Porodisculus pendulus), Botrytis In particular Botrytis cinerea , Laminaria , in particular Laminaria, Lentinula , in particular Lentinula edoles , and Monilinia , in particular Monilinia fructitiana ( Monilinia fructigena ).

A preferred microorganism for the process according to the invention is, for example, a Shijoilum combinum available from public deposits such as:

DSM-1024, DSM-1025, DSM-1026, DSM-11223

ATCC Number: 26892, ATCC Number: 62873, ATCC Number: 38229,

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

ATCC 占 Number: MYA-1128, ATCC 占 Number: 42093, ATCC 占 Number: 18246,

ATCC 占 Number: MYA-1124, ATCC 占 Number: 38230, ATCC 占 Number: 26889,

ATCC 占 Number: 26262, ATCC 占 Number: 52398, ATCC 占 Number: MYA-1123.

In another embodiment, the present invention is directed to a method of producing a polymer as described above by culturing any of the genetically engineered microorganisms described above.

The genetically engineered microorganisms according to the present invention can be obtained by the method described in, for example, 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. RTI ID = 0.0 > (1994-1998). ≪ / RTI > Further examples of the construction of genetically engineered microorganisms are disclosed in the experimental part.

Methods of culturing microorganisms, such as fermentation processes, are known in the art and are described and exemplified herein (Kumari, Bioresource Technol (2008), 99: 1036-1043; Reyes, J Natural Studies (2009), 7 -June). According to the present invention, such a method allows each microorganism to grow and produce the desired [beta] -glucan described and exemplified herein. Suitable media may include, for example, Reyes, coconut wattle described in the cited text. In addition, as is known in the art, there are several media that are particularly suitable for particular microorganisms.

For example, in accordance with the present invention, also suitable media for cultivating the callus is a CYM medium (25 g agar (Difco) per liter of H ₂ O, 20 g glucose (Sigma), 2 g tryptic case peptone ), 2 g yeast extract (Difco), 0.5 g MgSO ₄ x 7 H ₂ O (Roth), 0.5 g KH ₂ PO ₄ and 1 g K ₂ HPO ₄ (both from Riedel-de Haen) 30 g glucose (Sigma), 3 g yeast extract (Difco), 1 g KH ₂ PO ₄ (Riedel-de Haen), 0.5 g MgSO ₄ per liter of H ₂ O described and exemplified herein ₄ x 7 H ₂ O (Roth) (which is particularly useful for liquid cultures).

According to the present invention, the term "average molecular weight of about 0,3" means that on average about three per 10 β-D- (1-3) -glycopyranosyl units are linked to a single β- 6) It can mean that it is connected. In this context, the term "about" means that the average fraction is in the range of 0.1 to 0.5, preferably 0.2 to 0.4, more preferably 0.25 to 0.35, more preferably 0.25 to 0.33, still more preferably 0.27 to 0.33, May be within the range of 0.3 to 0.33. It can also be 0.3 or 0.33. Zymoylphenol, scleroglucan, penluan, cenerian, laminarin, lentinan and fleurans all have an average fractional map of 0.25 to 0.33; For example, scleroglucans and sipo-pilans have an average fractional map of 0.3 to 0.33 (Survase, cited text; Novak, cited text). The average molecular weight of? -glucan can be determined by methods known in the art, for example, by cyclic oxidation analysis, methylation sugar analysis and NMR (Brigand, Industrial Gums, Academic Press, New York / USA ), 461-472).

In one embodiment of the present invention, the polymer to be produced is selected from the group consisting of zymo-fylenes, scleroglucans, penluans, cinerians, laminarins, lentanans and fleurans. For example, the polymer may be a zymo-filan or a scleroglucan, especially a zymo-filan.

Recovery of the polymer from the fermentation product can be accomplished by a number of conventional techniques known in the art of biotechnology, such as precipitation and centrifugation.

Many processes for the production of? -glucan include 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 the production process, that is, Xeno is carried out by batch fermentation, agitation and aeration of the fungal mycelium compom. The medium mainly comprises glucose, yeast extract, potassium dihydrogenphosphate, magnesium sulfate and water. EP 271 907 A2 describes a method for isolating polysaccharides, wherein the culture suspension is first centrifuged and the polysaccharides are precipitated from the supernatant into isopropanol. The second method involves ultrafiltration of the resulting solution followed by pressure filtration, and details of this method have not been disclosed. "Udo Rau, Biopolymers, Editor A. Steinbuechel, Volume 6, pages 63-79", Udo Rau, "Biosynthese, Produktion und Eigenschaften von extrazellulaeren Pilz-Glucanen", Habilitationsschrift, Technical University of Brunswick, , WILEY-VCH Publishers, New York, 2002 "describes the preparation of zymo-pilans by continuous or batch fermentation. "GIT Fachzeitung Labor 12/92, pages 1233-1238" describes the continuous preparation of branched < RTI ID = 0.0 > ss-1,3-glucans < / RTI > WO 03/016545 A2 discloses a continuous process for the preparation of scleroglucans using sclerotium roulsiec.

In addition, for economic reasons, the concentration of aqueous β-glucan aqueous solution should be as high as possible to ensure as little transport effort as possible to transport the aqueous glucan solution from the production site to the point of use. For this purpose, the [beta] -glucan solution is conventionally concentrated by drying, lyophilization and / or precipitation before it is transported to reduce its weight.

The genetically engineered microorganism according to the present invention is at least 1.5 times, more preferably at least 1.8 times more, more preferably at least 2.0 times more, most preferably at least 2.2 times as much as the corresponding untreated control microorganism It is possible to produce many? -Glucan polymers. In this context, for example, the production of a 1.5-fold "more" -glucan polymer is preferred because the genetically engineered microorganism is able to produce a < RTI ID = 0.0 >Gt; beta-glucan polymer < / RTI > Alternatively, for example, the production of a 1.5-fold "more" [beta] -glucan polymer may allow the genetically engineered microorganism to grow the same amount of the [beta] -glucan polymer as the corresponding untreated control organism under the same conditions, It can mean faster production. The amount of the produced? -Glucan polymer is known in the art and can be determined by the methods described herein.

Experimental Part

Table 1. Strain used

The strain of Example 1 and culture conditions

(Dons JJM et al. (1979)) were grown for 7 days in the dark at 25 ° C in minimal medium (MM) agar (1.5% . Characterization of the genome of the basidiomycete Schizophyllum commune . Biochimica et Biophysica Acta 563 : 100-112). One quarter of the colonies were homogenized in 50 ml of liquid MM for 30 seconds at full speed in a Waring Blender. 50 ml of the homogenate was incubated in the dark at 250 < 0 > C and 200 rpm in a 250 ml Erlenmeyer flask. 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 containing 0.2 g wet weight mycelium was calculated. This volume was used to inoculate 100 ml of liquid shake culture with MM and 250 ml of Rendermeyer flask. The cultures were grown in triplicate at 25 ° C and 200 rpm in the dark in triplicate.

The strain of Example 2 and culture conditions

Colonies of the nucleated strains H4-8 and [Delta] hom2 [ Delta] hom2 were grown in MM agar plates in the dark for 7 days from spot inoculations. A 2x3 cm < 2 > fragment was cut from the middle section of these colonies. The agar was removed as much as possible and the mycelium was suspended in 15 ml of DHSV medium (33 g ℓ ^-1 glucose monohydrate, 0.5 g ℓ ^-1 MgSO ₄揃 7H ₂ O, 0.9 g ℓ ^-1 KH ₂ PO ₄ , 0.1 g ℓ ^{- 1} K ₂ HPO ₄ , 0.5 g ℓ ^-1 citric acid, 50 g ℓ ^-1 urea, pH 5.8 supplemented by spore elements: 20 mg l ^-1 FeSO ₄ .7H ₂ O, 50 mg l ^-1 Titriplex III (Merck Millipore), 1 mg l ^-1 ZnSO ₄ .7H ₂ O, 0.3 mg l ^-1 MnSO ₄ .4H ₂ O, 3 mg l ^-1 H ₃ BO ₃ , 2 mg l ^-1 CoCl ₂ .6H ₂ O , 0.1 mg l ^-1 CuCl ₂ .2H ₂ O, 0.2 mg l ^-1 NiCl ₂ .6H ₂ O, 0.3 mg l ^-1 Na ₂ MoO ₄ .2H ₂ O and vitamins: 0.0105 mg l ^-1 biotin, 0.0045 mg 1 ^- folic acid, 2.31 mg l ^-1 -1,4-aminobenzoic acid, 0.18 mg l ^-1 riboflavin, 0.825 mg l ^-1 calcium pantothenate, 1.8 mg l ^-1 nicotinamide, 0.135 mg l ^-1 pyridoxine-HCl, 4.2 ㎎ ℓ ^-1 myo-inositol and, at 6,000 rpm for one minute at 1.605 ㎎ ℓ ^-1 thiamine -HCl) using a T 25 digital ULTRA-TURRAX (IKA) Homogenized. Twenty-five ml of the pre-culture per strain was inoculated with 2.5 ml of homogenate. For this, 50 ml DHSV medium was added to 100 ml erlenmeyer. The cultures were grown in the dark at 30 < 0 > C and 180 rpm for 72 h. The pre-culture was centrifuged at 9,935 g for 10 min and the pellet of each strain was collected. The supernatant was collected, and glucose and glucan were measured as described below. The pooled pellet was again homogenized in 25 ml DHSV (see above). 100 ml of Erlenmeyer was used to inoculate 2.5 ml of the homogenate in 50 ml of DHSV culture. The cultures were grown in shadows three times at 30 ° C and 180 rpm for 3, 8 or 10 days.

The viscosity of the medium of Example 1

The culture medium was collected by centrifugation at 5,000 g for 10 minutes or by filtration on miraculous. Formic acid (4 g l- ¹ final concentration) was added to stabilize the medium and measure the viscosity. For this, a 10 mL BD Plastipak syringe with a Greiner Bio One blue pipette tip (1000 μl) attached to the nozzle was filled with culture medium. The time taken for 8 ml of fluid to flow out of the syringe (the upper level of the medium from the 10 ml marker position to the 2 ml marker position) was used as a measure of viscosity. This procedure was related to the measurement by HAAKE Rheostress 1 rotary rheometer 1 (Thermo Fischer). Measurements are correlated with Pearson correlation coefficients greater than 0.95.

Determination of glucan, urea, ethanol and biomass of Examples 1 and / or 2

To confirm SPG production, 10 ml of culture was pipetted into a 50 ml Falcon tube and stabilized with 3 g L- ¹ ACTICIDE BW20 (Thor) and incubated with Penicillium funiculosum (Erbsloeh) Lt; ⁰ > C for 24 h with 10 ml < ¹ > Submers [beta] -glucanase. After centrifugation at 3,400 g for 30 min, the ethanol and glucose concentrations in the supernatant were measured using an HPLC cation exchanger (Aminex HPX-87H, Bio-Rad) with 5 mM H ₂ SO ₄ (Roth) and 0.5 ml min ^-1 At a flow rate of 30 캜. The concentration of urea in the culture medium was also determined by HPLC. For this purpose, a fast carbohydrate column, 100 × 7.8 mm (Biorad), was used as the eluent with water and 1 ml min ^-1 flow rate at 70 ° C.

The pellet obtained after centrifugation (see above) for 30 min at 3,400 g was resuspended in 30 ml H ₂ O and vigorously shaken to confirm the biomass produced in the culture. After centrifugation at 3,400 g for 30 min, the pellet was resuspended again in 30 ml H ₂ O by shaking, placed on Whatman filter paper, rinsed twice with 50 ml water, and then vacuum dried. The sample was further dried and weighed using a Mettler Toledo HB43-S Halogen Moisture Analyzer. The weight of the filter (identified before the mycelium was placed on the filter) was subtracted.

Example 1

The viscosities of liquid shake cultures of the wild-type H4-8xH4-8b nucleoside and its derivative Δ hom2Δhom2 strain were measured during the 10-day incubation period. The viscosity of the medium was higher in the Δ hom2Δhom2 strain compared to the wild type from day 5 and day 6, due to the reduced flow rate of the culture medium outside the syringe (FIG. 1) and by the rotational rheometer 1 (FIG. 2) Respectively. The viscosity of the medium was related to SPG production by glucan analysis. To this end, the culture was applied to disruption by glucanase and the resulting glucose was quantitated by HPLC analysis (FIG. 3). Glucose release by glucanase activity was detected after 8 days and 10 days of incubation of the Δ hom2Δhom2 strain and the wild type strain, respectively. On day 10, glucose levels were 5-fold higher in the Δ hom2Δhom2 strain. Taken together, the Δ hom2Δhom2 strain produces more SPG as shown by viscosity and glucanase experiments.

The viscosity of the medium was also measured for the strains DELTA WC2 DELTA WC2 and DELTA fst4 DELTA fst4 and for the two complementary DELTA hom2 DELTA hom2 strains (FIGS. 4 and 5). The viscosity of the medium for all four strains was not significantly different from that of the wild type. Taken together, these data confirm that the absence of Hom2 increases sipoylpran production. In contrast, the absence of Fst4 and WC2 does not affect SPG production, despite the fact that these strains can not form fruiting bodies like the Δ hom2Δhom2 strain.

Example 2

The viscosities, glucans, glucose (added as a carbon source), urea, ethanol and biomass were quantified in a 3-, 8-, and 10-day old culture of Δ hom2Δhom2 and wild- 8). On days 8 and 10, the culture medium of the strain Δ hom2Δhom2 was more viscous than in the wild type, which is measured by a rheometer (FIG. 6). The amount of glucose released by glucanase after 10 days of culture was significantly higher in the Δ hom2Δhom2 strain compared to the wild type strain (FIG. 7). The Δ hom2Δhom2 strain showed less ethanol production on day 8 (twice the wild type), and this metabolite was even absent after 10 days (FIG. 8). In contrast, the wild type had more than doubled the amount of ethanol from day 8 to 10 (FIG. 8). On day 10, glucose and urea were completely consumed in the case of the Δ hom2Δhom2 nucleoside but not in the wild type (FIG. 8). The yield of SPG per biomass was 20% higher in the Δ hom2Δhom2 strain than in the wild type (FIG. 9). Moreover, the Δ hom2Δhom2 strain produced 3.7 times more SPG per glucose than the wild type. We conclude that the production of SPG is superior to that of the parents of Sijo pylomicum Δ hom2 .

List of drawings:

Figure 1. Viscosity of the culture medium of liquid shake culture of wild-type and Δhom2Δhom2 dinuclear cells of Shiso Philomachique, measured by flow rate through a syringe.

Figure 2. Viscosity (mPa · s) of the culture medium of liquid shake culture of the wild-type and Δhom2Δhom2 dinuclear body of Shijo Philomicum. The viscosity was measured with a rheometer using the same sample as in Fig.

Figure 3. Glucan analysis of culture media from liquid cultures of wild-type and Δhom2Δhom2 dinuclear cells of Shijo philomicum. Glucans were quantified by measuring the glucose release resulting from the glucanase treatment. Samples are the same as the samples of Figs. 1 and 2.

Figure 4. SPG production per gram of biomass in 10-day-old liquid cultures of the dinuclear wild-type strain H4-8 × H4-8b and the binuclear knockout strains Δhom2Δhom2 , ΔWC2ΔWC2 , and Δfst4Δfst4 .

Figure 5. Of the 10-day-old liquid cultures of the dinuclear wild-type strain H4-8xH4-8b, the dinuclear knockout strain Δ hom2Δhom2 , and the two complementary Δ hom2Δhom2 strains ( hom2 reintroduced into one of the parent kernels) Production of SPG per gram of biomass.

Figure 6. Viscosity (mPa · s) of the culture medium of liquid shake culture of the wild-type and Δhom2Δhom2 dinuclear body of Shijo Philomicum. The viscosity was measured with a rheometer.

Figure 7. Glucan analysis of culture media from liquid cultures of wild-type and [Delta] hom2 [ Delta] hom2 heterozygotes of Zygoophilum comneum . Glucans were quantified by measuring the glucose release resulting from the glucanase treatment. Samples were taken from the same culture as in Fig.

Figure 8. Glucose, urea and ethanol levels in the culture medium of liquid shake cultures of wild-type and Δhom2Δhom2 dinuclear cells of the zyphophyllum combos . The sample is the same as the sample of Fig.

Figure 9. SPG yield (grams / gram) per biomass of the dinuclear wild-type strain and its derivative Δ hom2Δhom2 strain of the zymophilus comneum and the yield of SPG per glucose (grams / gram).

SEQ ID NO: 1 is the polynucleotide sequence of the hom2 gene from the epidermal stratum comneum used in the experimental part.

SEQ ID NO: 2 is the polynucleotide sequence of the hom2 gene from the pseudomonas pneumoniae complex which is slightly different from that disclosed in SEQ ID NO: 1.

SEQ ID NO: 3 is the polypeptide sequence of the hom2 gene from the zypohilum combo translated from SEQ ID NO: 1.

SEQ ID NO: 4 is the polypeptide sequence of the hom2 gene from the zypohilum combo translated from SEQ ID NO: 2.

references

                         SEQUENCE LISTING <110> Wintershall Holding GmbH   <120> Process for producing a beta-glucan polymer and genetically        modified microorganisms useful in this process <130> PF00000076102 &Lt; 150 > EP 14152338.1 <151> 2014-01-23 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 2166 <212> DNA <213> Schizophyllum commune <400> 1 atgcttccac gctccgccag cattccttct cccacccacg ctctttccca gccagcgcat 60 ccgcaacagg tccccccact gcccttttat ccggaccagc gctcctttga gcccctcgaa 120 tacggtcacc gacacccgtc ccacttccac ggccatccct atcttcaaca ccccgggcag 180 cccatggaca tcctcgccga ctatcgcacc ttcttcccct accagcccaa cgaggtcaag 240 caccgtcggc gaaccaccgc tgtccagctc aaggtcctcg agggcatctt caagacggag 300 acgaagccga atgctgcgct gcggaacaag cttgccgtcc agctggaaat gaccgcgaga 360 ggtgtacagg tctggtttca gaaccgacgc gcgaaggaaa agctgaaggc cagcaaggca 420 agcaaggcaa aagaggagaa gagcaacgcc gataaggccg acgcatcccc acccagtccc 480 tccaaccctc ccacgggagg ttcgtcctcg tcgacgacga cgcccgcggc cgaagagtct 540 aagtcggacc agtcggctgc cactactcca cccaccacac caccctcgaa acccgactcg 600 ccccccgact cgtcggcgat cagcccacct gcgttgacca tcaacgtagc gccatcggag 660 cccccgtctt tgcaatctcc ttggcaagcc cctgcatctg gcggttctcc cgcccagcac 720 accctgccgg tcgcccccgt cccatcggtc gacgacgcgg cgctcctagc ccaacggcgc 780 ggctcgctgc ccgcccacct ctactcgacc gtcgatccct cgtccgacct gctctctgtg 840 caccatgacc agttcgcccg gcggcgatcg gtcgacgcca gcctgcagcg gctgccgtac 900 aacccgtacg cgcctgtcgc gcgcgcgcgg aacggcatgc actccgggct gaacccgcac 960 cgcggtcctg ctcagcgggg gctctgccca ccgatggcgc gcgcaatatc catggacgcg 1020 cggcgcttct cagtggacgt cgtgccctcc gcgcaacgca tgtactcgcg cccatctccc 1080 cagcttccct acagccgccc ctcacttccc aacagctccc tcacatccct tcccaacggc 1140 tcccgcccct ccctgcccaa cggcacaatg tacgcctata cacagcgtcc agtaccgcct 1200 tcacccatcc ccgggcccct ccccgcaccc aacttctcct tcggtgcgcc catgcctccg 1260 gccgagtccg agcaggactc gccacgacgc gaggacttcg acgggcatcc gcgcgacttc 1320 gacgggcagt cgctcgcctc ctcgtacgcc gcgtcctccc gcttcggctc cctggcgtcc 1380 atcagcacga ccgacagcca gcacacgtac tactcggacg tgacggaccc ggcggattac 1440 gcgctccgtc gcgcatctat gacctcgaac cagttcctgg ggatgatgag caaccttgat 1500 gtgagcgata acagcgggga gcagtgctat tcagggaagc aggcggccta tccaggggcg 1560 cagggcggta tgtatggggc ggcgcaggct cagcagcagc aacaggccgc gagcgaggcg 1620 catgtggctt accagacctc ggctgaggtt gcatacccct ccccggcccc gacgttgtcg 1680 ccgaaggata atgcgcttgg gatggggcct gcgcacatgc agcgagcgca gtctactatg 1740 cagagcgagc agcagcaggc gtacgagcaa caacaacagc aggtagcata cgaccaatcg 1800 cgctcgtacg agcagcagca gccatctgtc cccaccaatc agtcttcttg cagcccgccg 1860 gcgcctact accccgaagg tagcctgcag gtgcagtacc cggacatgta cgagacgggg 1920 atctcgccct ctgggcacta cgtacctgcc acttcgtact ccgtcccggc atacccgctg 1980 gacgacaact acatgggtgg ctatactgca accgacgcag ggtatgcggc caacgaggcg 2040 ggctacggcg tcaacgacgc gagctcgcgg ctcgagttcg cccacccgcc tgactggagc 2100 acggggtact cacccgtcgc ggagactgca ccatgcgact tgggcgcgta cgtgcggtat 2160 cagtaa 2166 <210> 2 <211> 2172 <212> DNA <213> Schizophyllum commune <400> 2 atgcttccac gctccgccag cattccttct cccacccacg ctctttccca gccagcgcat 60 ccgcaacagg tccccccacc gcccttttat ccggaccagc gctcctttga gcccctcgaa 120 tacggtcacc gacacccgtc ccacttccac ggccatccct atcttcaaca ccccgggcag 180 cccatggaca tcctcgccga ctatcgcacc ttcttcccct atcagcccaa cgaggtcaag 240 caccgtcggc gtaccaccgc tgtccagctc aaggtcctcg agggcatctt caagacggag 300 acgaagccga atgctgcgct gcggaacaag cttgccgtcc agctggaaat gaccgcgaga 360 ggtgtacagg tctggtttca gaaccgacgc gcgaaggaaa agctgaaggc cagcaaggca 420 agcaaggcaa aagaggagaa gagcaacgcc gataaggccg acgcatcccc acccagtccc 480 tccaaccctc ccacgggagg ttcgtcctcg tcgacgacga cgcccgcggc cgaagagtct 540 aagtcggacc agtcggctgc cactactcca cccaccacac caccctcgaa acccgactcg 600 ccccccgact cgtcggcgat cagcccacct gcgttgacca tcaacgtagc gccatcggag 660 cccccgtctt tgcaatctcc ttggcaagcc cctgcatctg gcggttctcc cgcccagcac 720 accctgccgg tcgcccccgt cccatcggtc gacgacgcgg cgctcctagc ccaacggcgc 780 ggctcgctgc ccgcccacct ctactcgacc gtcgatccct cgtccgacct gctctctgtg 840 caccatgacc agttcgcccg gcggcgatcg gtcgacgcca gcctgcagcg gctgccgtac 900 aacccgtacg cgcctgtcgc gcgcgcgcgg aacggcatgc actctggcct gaacccgcac 960 cgcggtcctg ctcagcgggg gctctgccca ccgatggcgc gcgcaatatc catggacgcg 1020 cggcgcttct cggtggacgt cgtgccctcg gcgcaacgca tgtactcgcg cccatctccc 1080 cagcttccct acagccgtcc ctcactcccc aacggctccc tcacatccct tcccaacggt 1140 tcccgcccct ccctgcctaa cggcacaatg tacgcctata cgcagcgtcc agtaccgccc 1200 tcacccatcc ccgggcccct ccccgcaccc aacttctcct tcggcgcgcc catgcccccg 1260 gccgagtccg agcaggactc gccgcggcgc gaggacttcg acgggcatcc gcgcgacttc 1320 gacgggcagt cgctcgcctc ctcctacgcc gcgtcctccc gcttcggctc cctggcgtcc 1380 atcagcacga ccgacagcca acacacgtac tactcggacg tgacggaccc ggcggattac 1440 gcgctccggc gcgcatctat gacctcgaac cagttcctgg ggatgatgag caaccttgat 1500 gtgagcgata acagcggaga gcagtgctat tcagggaagc aagcgggcta tccaggggcg 1560 caggggggta tgtatggggc ggcgcaggct cagcagcagc agcagacctc gggcgaagcg 1620 gctcatgcgg tgtatacctc cgaggcggcc catgctgcgt acccttctcc ggcgccgacg 1680 ctttcgccga aggacaatgc gcttgggatg ggccctgctc acatgcagcg agcgcagtct 1740 actatgcaga gcgagcagca gcaggcgtac gagcaacaac aacagcaaca ggcatacgac 1800 caatcgcgct cgtacgagca gcagcagcca tctgtccccg ccaatcagtc ctcttgcagc 1860 ccgccggacg cctactaccc cgaaggtagc ctgcaggtgc agtacccgga catgtacgag 1920 acggggatct cgccctctgg gcactacgtc cctgccacgt catactctgt cccggcgtac 1980 ccgctggacg acaactacat gggtggctat actgcaaccg acgctggcta cgcagccaac 2040 gatgcgggct acggcgtcaa cgacgcgagc tcgcggctcg agttcgccca cccgcccgac 2100 tggagcacgg ggtactcacc cgtcgcggag actgcgccgt gcgacttggg cgcgtacgtg 2160 cggtaccagt aa 2172 <210> 3 <211> 721 <212> PRT <213> Schizophyllum commune <400> 3 Met Leu Pro Arg Ser Ala Ser Ile Pro Ser Pro Thr His Ala Leu Ser 1 5 10 15 Gln Pro Ala His Pro Gln Gln Val Pro Pro Leu Pro Phe Tyr Pro Asp             20 25 30 Gln Arg Ser Phe Glu Pro Leu Glu Tyr Gly His Arg His Pro Ser His         35 40 45 Phe His Gly His Pro Tyr Leu Gln His Pro Gly Gln Pro Met Asp Ile     50 55 60 Leu Ala Asp Tyr Arg Thr Phe Phe Pro Tyr Gln Pro Asn Glu Val Lys 65 70 75 80 His Arg Arg Arg Thr Thr Ala Val Gln Leu Lys Val Leu Glu Gly Ile                 85 90 95 Phe Lys Thr Glu Thr Lys Pro Asn Ala Ala Leu Arg Asn Lys Leu Ala             100 105 110 Val Gln Leu Glu Met Thr Ala Arg Gly Val Gln Val Trp Phe Gln Asn         115 120 125 Arg Arg Ala Lys Glu Lys Leu Lys Ala Ser Lys Ala Ser Lys Ala Lys     130 135 140 Glu Glu Lys Ser Asn Ala Asp Lys Ala Asp Ala Ser Pro Pro Ser Pro 145 150 155 160 Ser Asn Pro Pro Thr Gly Gly Ser Ser Ser Thr Thr Thr Pro Ala                 165 170 175 Ala Glu Glu Ser Lys Ser Asp Gln Ser Ala Ala Thr Thr Pro Pro Thr             180 185 190 Thr Pro Pro Ser Lys Pro Asp Ser Pro Pro Asp Ser Ser Ala Ile Ser         195 200 205 Pro Pro Ala Leu Thr Ile Asn Val Ala Pro Ser Glu Pro Pro Ser Leu     210 215 220 Gln Ser Pro Trp Gln Ala Pro Ala Ser Gly Gly Ser Pro Ala Gln His 225 230 235 240 Thr Leu Pro Val Ala Pro Val Val Ser Val Asp Ala Ala Leu Leu                 245 250 255 Ala Gln Arg Arg Gly Ser Leu Pro Ala His Leu Tyr Ser Thr Val Asp             260 265 270 Pro Ser Ser Asp Leu Leu Ser Val His His His Asp Gln Phe Ala Arg Arg         275 280 285 Arg Ser Val Asp Ala Ser Leu Gln Arg Leu Pro Tyr Asn Pro Tyr Ala     290 295 300 Pro Val Ala Arg Ala Arg Asn Gly Met His Ser Gly Leu Asn Pro His 305 310 315 320 Arg Gly Pro Ala Gln Arg Gly Leu Cys Pro Pro Met Ala Arg Ala Ile                 325 330 335 Ser Met Asp Ala Arg Arg Phe Ser Val Asp Val Val Ser Ser Ala Gln             340 345 350 Arg Met Tyr Ser Arg Pro Ser Pro Gln Leu Pro Tyr Ser Arg Pro Ser         355 360 365 Leu Pro Asn Ser Ser Leu Thr Ser Leu Pro Asn Gly Ser Arg Pro Ser     370 375 380 Leu Pro Asn Gly Thr Met Tyr Ala Tyr Thr Gln Arg Pro Val Pro Pro 385 390 395 400 Ser Pro Ile Pro Gly Pro Leu Pro Ala Pro Asn Phe Ser Phe Gly Ala                 405 410 415 Pro Met Pro Pro Ala Glu Ser Glu Gln Asp Ser Pro Arg Arg Glu Asp             420 425 430 Phe Asp Gly His Pro Arg Asp Phe Asp Gly Gln Ser Leu Ala Ser Ser         435 440 445 Tyr Ala Ala Ser Ser Phe Gly Ser Leu Ala Ser Ser Thr Thr     450 455 460 Asp Ser Gln His Thr Tyr Tyr Ser Asp Val Thr Asp Pro Ala Asp Tyr 465 470 475 480 Ala Leu Arg Arg Ala Ser Met Thr Ser Asn Gln Phe Leu Gly Met Met                 485 490 495 Ser Asn Leu Asp Val Ser Asp Asn Ser Gly Glu Gln Cys Tyr Ser Gly             500 505 510 Lys Gln Ala Tyr Pro Gly Ala Gln Gly Gly Met Tyr Gly Ala Ala         515 520 525 Gln Ala Gln Gln Gln Gln Gln Ala Ala Ser Glu Ala His Val Ala Tyr     530 535 540 Gln Thr Ser Ala Glu Val Ala Tyr Pro Ser Pro Ala Pro Thr Leu Ser 545 550 555 560 Pro Lys Asp Asn Ala Leu Gly Met Gly Pro Ala His Met Gln Arg Ala                 565 570 575 Gln Ser Thr Met Gln Ser Glu GIn Gln Gln Ala Tyr Glu Gln Gln Gln             580 585 590 Gln Gln Val Ala Tyr Asp Gln Ser Ser Ser Tyr Glu Gln Gln Gln Pro         595 600 605 Ser Val Pro Thr Asn Gln Ser Ser Cys Ser Pro Pro Asp Ala Tyr Tyr     610 615 620 Pro Glu Gly Ser Leu Gln Val Gln Tyr Pro Asp Met Tyr Glu Thr Gly 625 630 635 640 Ile Ser Pro Ser Gly His Tyr Val Pro Ala Thr Ser Tyr Ser Val Pro                 645 650 655 Ala Tyr Pro Leu Asp Asp Asn Tyr Met Gly Gly Tyr Thr Ala Thr Asp             660 665 670 Ala Gly Tyr Ala Asn Aly Gly Aly Gly Tyr Gly Val Asn Asp Ala Ser         675 680 685 Ser Arg Leu Glu Phe Ala His Pro Pro Asp Trp Ser Thr Gly Tyr Ser     690 695 700 Pro Val Ala Glu Thr Ala Pro Cys Asp Leu Gly Ala Tyr Val Arg Tyr 705 710 715 720 Gln      <210> 4 <211> 723 <212> PRT <213> Schizophyllum commune <400> 4 Met Leu Pro Arg Ser Ala Ser Ile Pro Ser Pro Thr His Ala Leu Ser 1 5 10 15 Gln Pro Ala His Pro Gln Gln Val Pro Pro Pro Phe Tyr Pro Asp             20 25 30 Gln Arg Ser Phe Glu Pro Leu Glu Tyr Gly His Arg His Pro Ser His         35 40 45 Phe His Gly His Pro Tyr Leu Gln His Pro Gly Gln Pro Met Asp Ile     50 55 60 Leu Ala Asp Tyr Arg Thr Phe Phe Pro Tyr Gln Pro Asn Glu Val Lys 65 70 75 80 His Arg Arg Arg Thr Thr Ala Val Gln Leu Lys Val Leu Glu Gly Ile                 85 90 95 Phe Lys Thr Glu Thr Lys Pro Asn Ala Ala Leu Arg Asn Lys Leu Ala             100 105 110 Val Gln Leu Glu Met Thr Ala Arg Gly Val Gln Val Trp Phe Gln Asn         115 120 125 Arg Arg Ala Lys Glu Lys Leu Lys Ala Ser Lys Ala Ser Lys Ala Lys     130 135 140 Glu Glu Lys Ser Asn Ala Asp Lys Ala Asp Ala Ser Pro Pro Ser Pro 145 150 155 160 Ser Asn Pro Pro Thr Gly Gly Ser Ser Ser Thr Thr Thr Pro Ala                 165 170 175 Ala Glu Glu Ser Lys Ser Asp Gln Ser Ala Ala Thr Thr Pro Pro Thr             180 185 190 Thr Pro Pro Ser Lys Pro Asp Ser Pro Pro Asp Ser Ser Ala Ile Ser         195 200 205 Pro Pro Ala Leu Thr Ile Asn Val Ala Pro Ser Glu Pro Pro Ser Leu     210 215 220 Gln Ser Pro Trp Gln Ala Pro Ala Ser Gly Gly Ser Pro Ala Gln His 225 230 235 240 Thr Leu Pro Val Ala Pro Val Val Ser Val Asp Ala Ala Leu Leu                 245 250 255 Ala Gln Arg Arg Gly Ser Leu Pro Ala His Leu Tyr Ser Thr Val Asp             260 265 270 Pro Ser Ser Asp Leu Leu Ser Val His His His Asp Gln Phe Ala Arg Arg         275 280 285 Arg Ser Val Asp Ala Ser Leu Gln Arg Leu Pro Tyr Asn Pro Tyr Ala     290 295 300 Pro Val Ala Arg Ala Arg Asn Gly Met His Ser Gly Leu Asn Pro His 305 310 315 320 Arg Gly Pro Ala Gln Arg Gly Leu Cys Pro Pro Met Ala Arg Ala Ile                 325 330 335 Ser Met Asp Ala Arg Arg Phe Ser Val Asp Val Val Ser Ser Ala Gln             340 345 350 Arg Met Tyr Ser Arg Pro Ser Pro Gln Leu Pro Tyr Ser Arg Pro Ser         355 360 365 Leu Pro Asn Gly Ser Leu Thr Ser Leu Pro Asn Gly Ser Arg Pro Ser     370 375 380 Leu Pro Asn Gly Thr Met Tyr Ala Tyr Thr Gln Arg Pro Val Pro Pro 385 390 395 400 Ser Pro Ile Pro Gly Pro Leu Pro Ala Pro Asn Phe Ser Phe Gly Ala                 405 410 415 Pro Met Pro Pro Ala Glu Ser Glu Gln Asp Ser Pro Arg Arg Glu Asp             420 425 430 Phe Asp Gly His Pro Arg Asp Phe Asp Gly Gln Ser Leu Ala Ser Ser         435 440 445 Tyr Ala Ala Ser Ser Phe Gly Ser Leu Ala Ser Ser Thr Thr     450 455 460 Asp Ser Gln His Thr Tyr Tyr Ser Asp Val Thr Asp Pro Ala Asp Tyr 465 470 475 480 Ala Leu Arg Arg Ala Ser Met Thr Ser Asn Gln Phe Leu Gly Met Met                 485 490 495 Ser Asn Leu Asp Val Ser Asp Asn Ser Gly Glu Gln Cys Tyr Ser Gly             500 505 510 Lys Gln Ala Gly Tyr Pro Gly Ala Gln Gly Gly Met Tyr Gly Ala Ala         515 520 525 Gln Ala Gln Gln Gln Gln Gln Thr Ser Gly Glu Ala Ala His Ala Val     530 535 540 Tyr Thr Ser Glu Ala Ala His Ala Ala Tyr Pro Ser Pro Ala Pro Thr 545 550 555 560 Leu Ser Pro Lys Asp Asn Ala Leu Gly Met Gly Pro Ala His Met Gln                 565 570 575 Arg Ala Gln Ser Thr Met Gln Ser Glu Gln Gln Gln Ala Tyr Glu Gln             580 585 590 Gln Gln Gln Gln Gln Ala Tyr Asp Gln Ser Ser Ser Tyr Glu Gln Gln         595 600 605 Gln Pro Ser Val Pro Ala Asn Gln Ser Ser Cys Ser Pro Pro Asp Ala     610 615 620 Tyr Tyr Pro Glu Gly Ser Leu Gln Val Gln Tyr Pro Asp Met Tyr Glu 625 630 635 640 Thr Gly Ile Ser Pro Ser Gly His Tyr Val Pro Ala Thr Ser Tyr Ser                 645 650 655 Val Pro Ala Tyr Pro Leu Asp Asp Asn Tyr Met Gly Gly Tyr Thr Ala             660 665 670 Thr Asp Gly Tyr Aslan Ala Asn Asp Ala Gly Tyr Gly Val Asn Asp         675 680 685 Ala Ser Ser Arg Leu Glu Phe Ala His Pro Pro Asp Trp Ser Thr Gly     690 695 700 Tyr Ser Pro Val Ala Glu Thr Ala Pro Cys Asp Leu Gly Ala Tyr Val 705 710 715 720 Arg Tyr Gln             

Claims (5)

(1-3) -glycopyranosyl unit having a single? -D-glucopyranosyl unit linked to a linear main chain? -D-glucopyranosyl unit having an average molecular weight of about 0.3 (1-6) Of a linear backbone comprising the steps of:
(i) β-D- (1-3) -glucose having a single β-D-glucopyranosyl unit linked to a linear main chain β-D-glucopyranosyl unit having an average branching of about 0.3 (1-6) A genetically engineered microorganism capable of producing a polymer consisting of a linear main chain of a pyranosyl unit, wherein said microorganism is a microorganism that confers a reduced activity of the hom2 gene product compared to an untreated control microorganism of the same strain, Culturing under conditions permitting production of said polymer;
(ii) optionally recovering said polymer from the medium. The method of claim 1, wherein the polymer is selected from the group consisting of zymo-phylan, scleroglucan, penluan, cynthian, laminarin, lentanan, and furan. 2. The method of claim 1, wherein the reduced activity of the hom2 gene product is caused by gene knock-out of the hom2 gene. 4. The method according to claim 3, wherein the genetically engineered microorganism is a dinuclear chitosan. 5. The method of claim 4, wherein the nucleotide has a double green-out of the hom2 gene.

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