ERYTHRITOL PRODUCING SAPROTROPH

Abstract

The present invention pertains to a genetically modified saprotroph for the biotechnological production of erythritol and a method for the production of erythritol using said genetically modified saprotroph.

Claims

1. A genetically modified saprotroph comprising at least one gene encoding at least one membrane-bound alditol transporter, at least one gene encoding at least one erythrose reductase and at least one inactivated gene encoding mannitol 1-phosphate 5-dehydrogenase.

2. The genetically modified saprotroph according to claim 1, wherein the saprotroph is a filamentous fungus selected from the genera Hypocrea, Gibberella, Aspergillus and Penicillium.

3. The genetically modified saprotroph according to claim 1, wherein the saprotroph is Hypocrea jecorina (Trichoderma reesei).

4. The genetically modified saprotroph according to claim 1, wherein the at least one gene encoding at least one membrane-bound alditol transporter is fps1.

5. The genetically modified saprotroph according to claim 1, wherein the at least one gene encoding at least one erythrose reductase is err1.

6. The genetically modified saprotroph according to claim 1, wherein the at least one inactivated gene encoding mannitol 1-phosphate 5-dehydrogenase is mpdh.

7. The genetically modified saprotroph according to claim 1, wherein the saprotroph further comprises at least one gene encoding at least one transketolase, in particular tkl1.

8. The genetically modified saprotroph according to claim 1, wherein the saprotroph further comprises at least one gene encoding at least one transaldolase, in particular tal1.

9. The genetically modified saprotroph according to claim 1, wherein the saprotroph further comprises at least one inactivated gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1).

10. The genetically modified saprotroph according to claim 1, wherein the at least one gene encoding at least one membrane-bound alditol transporter, the at least one gene encoding at least one erythrose reductase, the at least one gene encoding at least one transketolase and/or the at least one gene encoding at least one transaldolase is overexpressed.

11. A method for the production of erythritol, comprising the steps: a) providing at least one genetically modified saprotroph according to claim 1, b) culturing the at least one genetically modified saprotroph provided in step a) in the presence a culture medium, so as to obtain erythritol in the culture medium, c) recovering erythritol from the culture medium.

12. The method according to claim 11, wherein the culture medium comprises lignocellulosic biomass, in particular straw, and/or at least one residue of dairy product production, in particular whey.

13. The method according to claim 11, wherein the culture medium comprises nitrate as a nitrogen source.

14. The method according to claim 11, wherein the culture medium comprises glycerol.

15. The method according to claim 11, wherein step b) is conducted until a concentration of erythritol in the culture medium of at least 1 g/L, preferably at least 1.5 g/L, preferably at least 2 g/L, preferably at least 2.5 g/L, preferably at least 3 g/L, is reached.

16. The method according to claim 11, wherein in step b) at least one substrate that causes osmotic stress is added to the culture medium after 14 hours of cultivation.

17. The method according to claim 16, wherein the substrate that causes osmotic stress is glycerol and/or sodium chloride.

18. Use of a genetically modified saprotroph according to claim 11 for the production of erythritol.

Description

[0127] The invention is further described by way of the following example and the accompanying figures.

[0128] FIG. 1 shows a comparison dry biomass of a T. reesei wild-type strain (WT), of a T. reesei strain (strain 1) overexpressing the alditol transporter gene fps1 and comprising an inactivated gene encoding mannitol 1-phosphate 5-dehydrogenase (mpdh) and of a T. reesei strain (strain 2) overexpressing the alditol transporter gene fps1 and the gene err1 encoding erythrose reductase as well as comprising an inactivated gene encoding mannitol 1-phosphate 5-dehydrogenase (mpdh) after 40 hours of cultivation.

[0129] FIG. 2 shows the concentration of glucose in the culture medium after 18, 28, 30, 32, 34, 36, 38 and 40 hours of cultivation of the wild-type strain (WT) and strains 1 and 2.

[0130] FIG. 3 shows the concentration of glycerol in the culture medium after 18, 28, 30, 32, 34, 36, 38 and 40 hours of cultivation of the wild-type strain (WT) and strains 1 and 2.

[0131] FIG. 4 shows the concentration of erythritol in the culture medium after 18, 28, 30, 32, 34, 36, 38 and 40 hours of cultivation of the wild-type strain (WT) and strains 1 and 2.

[0132] FIG. 5 shows a plasmid map of pKR_ReAsl1fps1 (see also SEQ ID No. 9).

[0133] FIG. 6 shows a plasmid map of pKR_Rpyr4err1 (see also SEQ ID No 0.10).

[0134] FIG. 7 shows a deletion cassette for deletion of mpdh (see also SEQ ID No. 11).

EXAMPLE

[0135] 1.1 Comparison of T. reesei Strains

[0136] For the characterization of the phenotype the following strains were used: [0137] the wild type strain QM6aΔtmus53, short-termed “WT”, [0138] QM6aΔtmus53 fps1(Reasl1)Δmpdh(hygR), short-termed “strain 1”, and [0139] QM6aΔtmus53 fps1(Reasl1)OEerr1(Repyr4)Δmpdh(hygR), short-termed “strain 2”. This strain has been deposited on May 25, 2020 in the Restricted CBS collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands by the depositor Technische Universität Wien (Gumpendorfer Straβe 1a, 1060 Vienna, Austria) under the stipulations of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The corresponding viability certificate has been emitted and the strain has been given accession number CBS 146708.

[0140] 1.2 Materials and Methods

[0141] 1.2.1 Generation of T. reesei Strains

[0142] The parental T. reesei strain used in the experiments is auxotroph for uridine (lack of functional pyr4), arginine (lack of functional asl1), histidine (lack of functional 80820 HisG) and is further resistant to hygromycin (hygR). The first transformation with the plasmid pKR_Reasl1fps1 (see FIG. 5) to restore the asl1 locus not only introduces the fps1 gene into the genome, but also makes the strain hygromycin sensible again. The err1 gene encoding erythrose reductase was introduced using the plasmid pKR_Repyr4err1 (see FIG. 6). The deletion of mpdh was done by transformation of a deletion cassette (see FIG. 7) to regain hygromycin resistance by homologous recombination. All transformations are performed as protoplast transformations. Typically, 30 to 50 μg of digested plasmid DNA (in 15 μl sterile double-distilled water) were used for transformation of 10.sup.7 protoplasts (in 200 μl). For selection for resistance against hygromycin B, 100 μl to 2 ml of the transformation reaction mixture was added to 10 ml melted, 50° C. warm malt extract (MEX) agar containing 1.2 M sorbitol. This mixture was poured into sterile petri dishes and incubated at 30° C. for 5 h after solidification. Subsequently, 10 ml melted, 50° C. MEX agar containing 1.2 M sorbitol and a double concentration of hygromycin B was poured on top of the protoplast-containing layer. Plates were incubated at 30° C. for 2 to 5 days until colonies were visible. For selection for prototrophy, 100 μl to 2 ml of the transformation reaction mixture was added to 20 ml melted, 50° C. warm minimal medium agar containing 1.2 M sorbitol and, in the case of asl1 back transformation, additionally 0.25 mM L-arginine. This mixture was poured into sterile petri dishes and after solidification was incubated at 30° C. for 3 to 7 days until colonies were visible.

[0143] 1.2.2 Cultivation of T. reesei and Induction of Erythritol Synthesis

[0144] In the experiment ammonium was used as nitrogen source and glucose as carbon source under osmotic stress. The total duration of cultivation was 40 hours. After 10 hours of cultivation for biomass production, sodium chloride was added to the medium in order to initiate osmotic stress. Culture broth samples have been taken at the time of this addition and next after 28 hours of total cultivation time and then every two hours until end of the cultivation. Samples were centrifuged to separate the mycelium from the supernatant.

[0145] Cultivation was performed with the wild-type strain (WT), QM6a_fm (strain 1) and QM6a_fem (strain 2) in 100 ml shake flasks containing 25 ml cultivation medium ((NH.sub.4).sub.2SO.sub.4 2.80 g/l, KH.sub.2PO.sub.4 4.00 g/l, MgSO.sub.4x7H.sub.2O 1.0 g/l, NaCl 0.5 g/l, peptone from Casein 0.1 g/l, Tween®80 0.5 g/l), supplemented with 1.5 ml/l trace element solution (FeSO.sub.4x7H.sub.2O 0.90 mM, MnSO.sub.4xH.sub.2O 0.50 mM, ZnSO.sub.4x7H.sub.2O 0.24 mM, CaCl.sub.2x7H.sub.2 0.68 mM). For inoculation 10.sup.9 conidiospores per liter were used. To all flasks 0.3 ml of 0.2 M histidine and 0.25 ml of 0.2 M uridine was added. After 10 hours 0.5 ml of 5 M NaCl was added. The agitation rate was 180 rpm, the temperature was 30° C.

[0146] After the cultivation the whole cultivation broth was filtered through miracloth, the mycelia were pressed dry with Whatman filter paper and dried at 80° C. overnight.

[0147] Glucose, glycerol, mannitol and erythritol concentrations were quantified by high-performance liquid chromatography (HPLC) (Shimadzu Prominence Series equipped with a RID-20A refractive-index detector). Culture supernatants were filtered through 0.20 μm membranes and measured by HPLC using a Rezex RCM-Monosaccharide column (300 mm×7.8 mm; Phenomenex, Torrance, CA, USA). The mobile phase was 30% acetonitrile (HPLC grade). For sample analysis, the column was eluted at 60° C. with 30% acetonitrile (HPLC grade) at a flow rate of 0.6 ml/min.

[0148] 1.3 Results

[0149] The biomass production at the end of the cultivation is nearly the same for all strains. WT produces slightly more biomass (FIG. 1). The glucose consumption is faster in strain 2 compared to WT and strain 1, which is deduced from concentration measurements in the respective supernatants (FIG. 2).

[0150] The glycerol concentration in the supernatant of strain 1 is always higher than in the WT due to the deletion of mannitol 1-phosphate 5-dehydrogenase (FIG. 3) since this deletion results in elimination of the production of mannitol and the strains produce more of the osmolyte glycerol. Whereas strain 1 shows a constant increase in glycerol production, strain 2 bearing the overexpressed err1 gene shows a fluctuating accumulation of glycerol over time (FIG. 3). Erythritol is only detectable in the supernatants of strains bearing the transporter FPS1 of Saccharomyces cerevisiae (FIG. 4). Investigations on the consumption of erythritol demonstrated that all strains, including WT, can grow on erythritol as sole carbon source (data not shown). The presence of erythritol in the supernatant seems to coincide with the secretion of glycerol and it is higher for strain 2 (FIG. 3 and FIG. 4).