Biotechnological method for the production of erythritol

Abstract

The present invention relates to a method for the biotechnological production of erythritol, in particular by cultivating at least one saprophytic fungus in a medium containing a nitrogen source and a high concentration of a carbon source.

Description

[Technical field]

[0001] The present invention relates to a method for the biotechnological production of erythritol, in particular by cultivating at least one saprophytic fungus in a medium containing a nitrogen source and a high concentration of a carbon source. [Background technology]

[0002] In recent years, due to people's lifestyles and increased intake of foods high in sugar, blood glucose-related diseases and disorders such as type 2 diabetes mellitus (DMT2) have been rapidly increasing. Today, it is believed that consuming low-glycaemic foods and avoiding excessive increases in blood glucose levels can reduce the risk of developing certain chronic diseases, and be beneficial for maintaining and improving health, as well as for treating and / or preventing many blood glucose-related diseases and disorders.

[0003] Erythritol is a naturally occurring 4-carbon sugar alcohol that is gaining importance in the food industry due to its unique properties and diverse range of applications. Erythritol is found in several fruits such as pears, grapes, and melons, mushrooms, alcoholic beverages (beer, wine, sake, etc.), and fermented foods such as soy sauce and miso, but also naturally occurs in human and animal biofluids such as lens tissue, serum, plasma, fetal fluid, and urine. Erythritol is easily absorbed in the upper intestine due to its small molecular weight, and therefore causes less indigestion than other sugar alcohols used in the food industry. Most of the ingested erythritol is not metabolized in the human body and is excreted unchanged in the urine without changing blood glucose or insulin levels (Regnat et al., Erythritol as sweetener-wherefrom and whereto?, 2018, Applied Microbiology and Biotechnology, Vol.102). Furthermore, erythritol is non-cariogenic, heat stable, crystallizes well, and is less hygroscopic than sucrose. Erythritol has a negative enthalpy of solution, which gives a cooling sensation in the mouth when ingested. A 10% (w / v) solution of erythritol is 60-80% as sweet as the same concentration of sucrose.

[0004] However, in contrast to other sugar alcohols such as sorbitol, xylitol, mannitol, lactitol, and maltitol, which have been established as sugar substitutes for many years, erythritol cannot currently be produced chemically in a commercially viable way. Production of erythritol from dialdehyde starch using nickel catalysts at high temperatures remains unsatisfactory in low yields (Moon et al. Biotechnological production of erythritol and its applications, 2010, Appl. Microbiol. Biotechnol., Vol.86).

[0005] In yeast and fungi, erythritol is produced via the so-called pentose phosphate pathway, which is synthesized from D-erythrose-4-phosphate via dephosphorylation of erythrose and its subsequent reduction. Based on this, several studies have investigated whether osmophilic yeasts such as Aureobasidium sp., Trichosporonoides sp., and Candida magnoliae are suitable for biotechnological production of erythritol (Ishizuka et al., Breeding of a mutant of Aureobasidium sp. with high erythritol production, 1989, J. Ferm. Bioeng., Vol. 68(5); US4939091A; US5962287 A; Oh et al., Increased erythritol production in fedbatch cultures of Torula sp. by controlling glucose concentration, 2001, JIM&B, Vol.26; Koh et al., Scale-up of erythritol production by an osmophilic mutant of Candida magnoliae, 2003, Biotechnol. Lett., Vol.25; Ryu et al., Optimization of erythritol production by Candida magnoliae in fed-batch culture, 2000, JIM&B, Vol.25). More recent studies have investigated the possibility of erythritol production by filamentous fungi (Jovanovic et al., 2013). However, the yields of erythritol obtained from the different strains were not satisfactory for industrial-scale production. Summary of the Invention [Problem to be solved by the invention]

[0006] There is therefore a need for a biotechnological method for producing erythritol with a high yield.The present invention overcomes the drawbacks of the prior art methods by the subject matter of the independent claims, in particular by the method for producing erythritol according to the invention. [Means for solving the problem]

[0007] The present invention relates in particular to a method for producing erythritol, comprising the steps of: a) culturing at least one saprophytic fungus in a medium containing a carbon source and a nitrogen source at a concentration of at least 40 g / L to obtain erythritol in the medium; b) recovering erythritol from the medium; The present invention relates to a method comprising the steps of:

[0008] In a preferred embodiment of the invention, the medium comprises a carbon source at a concentration of at least 50 g / L, preferably at least 60 g / L, preferably at least 70 g / L.

[0009] In a further preferred embodiment of the invention the medium comprises a carbon source at a concentration of at most 150 g / L, preferably at most 125 g / L, preferably at most 100 g / L, preferably at most 95 g / L, preferably at most 90 g / L.

[0010] According to a preferred embodiment of the present invention, the medium contains a carbon source at a concentration of 40 g / L to 200 g / L, preferably 45 to 175 g / L, preferably 50 to 150 g / L, preferably 55 to 125 g / L, preferably 60 to 100 g / L, preferably 65 to 95 g / L, preferably 70 to 90 g / L.

[0011] Preferably, the medium contains a carbon source at a concentration of 40 to 160 g / L, preferably 40 to 150 g / L, preferably 40 to 140 g / L, preferably 45 to 130 g / L, preferably 45 to 120 g / L, preferably 45 to 110 g / L, preferably 50 to 100 g / L, preferably 50 to 95 g / L, preferably 50 to 90 g / L, preferably 60 to 90 g / L, preferably 65 to 90 g / L, preferably 70 to 90 g / L.

[0012] In a further preferred embodiment of the invention the medium comprises a nitrogen source at a concentration of at least 50 mM, preferably at least 55 mM, preferably at least 60 mM, preferably at least 65 mM, preferably at least 70 mM.

[0013] Preferably, the medium comprises a nitrogen source at a concentration of at most 150 mM, preferably at most 140 mM, preferably at most 130 mM, preferably at most 120 mM, preferably at most 110 mM, preferably at most 100 mM.

[0014] Particularly preferably, the medium contains a nitrogen source at a concentration of 50 to 140 mM, preferably 55 to 130 mM, preferably 60 to 120 mM, preferably 65 to 110 mM, preferably 70 to 100 mM.

[0015] According to a preferred embodiment of the present invention, the medium is a synthetic medium. Preferably, the medium is not a synthetic medium.

[0016] In a further preferred embodiment of the invention, the medium comprises a hydrolysate obtained by hydrothermal treatment of a raw material containing cellulose, hemicellulose and / or starch.

[0017] Particularly preferably, the medium comprises a lignocellulose-containing hydrolysate.

[0018] In a further preferred embodiment of the invention, the hydrolysate, preferably a hydrolysate obtained by hydrothermal treatment of a raw material comprising cellulose, hemicellulose and / or starch, in particular a hydrolysate comprising lignocellulose, is derived from agro-industrial residues.

[0019] According to a preferred embodiment of the invention, the medium comprises a straw hydrolysate, in particular a wheat straw hydrolysate. In a further preferred embodiment, the medium comprises a bran hydrolysate. Preferably, the medium comprises a potato pulp hydrolysate.

[0020] In a preferred embodiment of the present invention, the carbon source is a monomeric or oligomeric C-5 and / or C-6 sugar or a mixture thereof. Preferably, the carbon source is a monomeric or oligomeric C-5 and / or C-6 sugar selected from arabinose, xylose, glucose, galactose, mannose and fructose. According to a further preferred embodiment, the carbon source is a mixture of monomeric and oligomeric C-5 and / or C-6 sugars, preferably a mixture of monomeric and oligomeric C-5 and / or C-6 sugars selected from arabinose, xylose, glucose, galactose, mannose and fructose.

[0021] In a further preferred embodiment, the carbon source is glucose or xylose. Particularly preferred, the carbon source is glucose. Preferably, the carbon source is xylose.

[0022] According to a preferred embodiment, the content of C-5 sugars in the medium amounts to at least 5%, preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% (based on the total carbohydrates in the medium).

[0023] Preferably, the content of C-6 sugars in the medium amounts to at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% (based on the total carbohydrates in the medium).

[0024] According to a preferred embodiment of the invention, the glucose content in the medium amounts to at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% (based on the total carbohydrates in the medium).

[0025] According to another preferred embodiment, the xylose content in the medium amounts to at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% (based on the total carbohydrates in the medium).

[0026] Preferably, the content of monomeric C-5 and C-6 sugars in the medium reaches at least 1%, preferably at least 2%, preferably at least 3%, preferably 4%, preferably at least 5%, preferably at least 6%, preferably at least 7%, preferably at least 8%, preferably at least 9%, preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% (based on the total carbohydrates in the medium).

[0027] According to a preferred embodiment of the invention, the content of oligomeric C-5 and C-6 sugars in the medium reaches at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% (based on the total carbohydrates in the medium).

[0028] According to a preferred embodiment of the present invention, the nitrogen source is selected from ammonium salts, nitrates, yeast extract and urea. Particularly preferably, the nitrogen source is an ammonium salt or urea. Preferably, the ammonium salt is selected from ammonium sulfate, ammonium nitrate, ammonium citrate, ammonium succinate, ammonium carbonate, ammonium oxalate and ammonium malate.

[0029] In a particularly preferred embodiment of the invention, the nitrogen source is urea.

[0030] Preferably, the medium comprises urea at a concentration of at least 8 mM, preferably at least 10 mM, preferably at least 15 mM, preferably at least 20 mM, preferably at least 25 mM, preferably at least 30 mM, preferably at least 35 mM, preferably at least 40 mM, preferably at least 45 mM, preferably at least 50 mM, preferably at least 55 mM, preferably at least 60 mM, preferably at least 65 mM, preferably at least 70 mM, preferably at least 80 mM.

[0031] According to a preferred embodiment of the invention, the culture medium comprises urea at a concentration of at most 160 mM, preferably at most 150 mM, preferably at most 140 mM, preferably at most 130 mM, preferably at most 120 mM, preferably at most 110 mM, preferably at most 100 mM, preferably at most 90 mM.

[0032] In a preferred embodiment, the medium contains urea at a concentration of 8 to 160 mM, preferably 10 to 150 mM, preferably 20 to 140 mM, preferably 30 to 130 mM, preferably 40 to 120 mM, preferably 50 to 110 mM, preferably 60 to 100 mM, preferably 65 to 100 mM, preferably 70 to 100 mM, and most preferably 70 to 90 mM.

[0033] According to a further preferred embodiment of the invention, the at least one saprophytic fungus is a filamentous fungus, preferably selected from the group consisting of Hypocrea, Trichoderma, Gibberella, Fusarium, Aspergillus and Penicillium.

[0034] In a preferred embodiment, the saprophytic fungus is selected from the group consisting of Hypocrea jecorina (Trichoderma reesei), red mold (Gibberella zeae), red mold of wheat (Fusarium graminearum), Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Penicillium chrysogenum. Particularly preferably, the saprophytic fungus is Hypocrea jecorina (Trichoderma reesei).

[0035] According to a preferred embodiment of the invention, the at least one saprophyte is a naturally occurring saprophyte, in particular not a genetically modified saprophyte.

[0036] In another preferred embodiment of the invention, the at least one saprophytic fungus is genetically modified.

[0037] In a particularly preferred embodiment of the invention, the genetically modified saprophytic fungus is Hypocrea jecorina (Trichoderma reesei). Preferably, the genetically modified saprophytic fungus is based on the Trichoderma reesei strain QM6aΔtmus53.

[0038] Preferably, the at least one genetically modified saprophytic fungus comprises 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.

[0039] In a further preferred embodiment of the present invention, the at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter is fps1, in particular codon-optimized fps1. Preferably, the at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter is fps1 from Saccharomyces cerevisiae, in particular codon-optimized fps1 from Saccharomyces cerevisiae.

[0040] In a further preferred embodiment, at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter comprises, in particular consists of, the nucleotide sequence of SEQ ID NO:1.

[0041] In a preferred embodiment of the present invention, at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 1. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 1.

[0042] Preferably, the membrane-bound alditol transporter of the recombinant saprophytic fungus comprises the amino acid sequence of SEQ ID NO:2, in particular consists of the amino acid sequence of SEQ ID NO:2.

[0043] Preferably, the recombinant saprophytic membrane-bound alditol transporter comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 2. In a further preferred embodiment, the recombinant saprophytic membrane-bound alditol transporter consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 2.

[0044] In another preferred embodiment of the present invention, the recombinant saprophytic membrane-bound alditol transporter comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 2 are replaced by other naturally occurring amino acids. In a particularly preferred embodiment, the recombinant saprophytic membrane-bound alditol transporter comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 2 are replaced by other naturally occurring amino acids.

[0045] In a preferred embodiment of the present invention, the at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase is err1, in particular a codon-optimized err1. Preferably, the at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase is err1, in particular a codon-optimized err1, from Trichoderma reesei, Aspergillus niger or Fusarium graminearum.

[0046] In a preferred embodiment, at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase comprises, in particular consists of, the nucleotide sequence of SEQ ID NO:3.

[0047] Preferably, the at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 3. In another preferred embodiment of the present invention, the at least one gene of the genetically modified saprophytic fungus encoding at least one gene encoding at least one erythrose reductase consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 3.

[0048] Preferably, the recombinant saprophytic fungal erythrose reductase comprises the amino acid sequence of SEQ ID NO:4, in particular consists of the amino acid sequence of SEQ ID NO:4.

[0049] In a preferred embodiment, the recombinant saprophytic erythrose reductase comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 4. Particularly preferably, the recombinant saprophytic erythrose reductase consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 4.

[0050] Preferably, the recombinant saprophytic erythrose reductase comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 4 are replaced by other naturally occurring amino acids. In a preferred embodiment of the present invention, the recombinant saprophytic erythrose reductase comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 4 are replaced by other naturally occurring amino acids.

[0051] In a preferred embodiment of the present invention, the at least one inactivated gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is mpdh.

[0052] Preferably, at least one gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO:9 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO:9 prior to inactivation.

[0053] Preferably, the at least one gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus comprised a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 9 before inactivation. In another preferred embodiment, the at least one gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus consisted of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 9 before inactivation.

[0054] Preferably, the mannitol-1-phosphate-5-dehydrogenase encoded by the nucleotide sequence of SEQ ID NO:9 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:10.

[0055] In a preferred embodiment, the mannitol-1-phosphate-5-dehydrogenase encoded by the nucleotide sequence of SEQ ID NO: 9 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 10. Particularly preferably, the mannitol-1-phosphate-5-dehydrogenase encoded by the nucleotide sequence of SEQ ID NO: 9 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 10.

[0056] Preferably, the mannitol-1-phosphate-5-dehydrogenase encoded by the nucleotide sequence of SEQ ID NO: 9 before inactivation comprises a sequence in which one, preferably two, preferably three, preferably five, preferably six, preferably seven, preferably eight, preferably nine, preferably ten amino acids in the amino acid sequence defined in SEQ ID NO: 10 are replaced by other naturally occurring amino acids. In a preferred embodiment of the present invention, the mannitol-1-phosphate-5-dehydrogenase encoded by the nucleotide sequence of SEQ ID NO: 9 before inactivation comprises a sequence in which at most ten, preferably at most nine, preferably at most eight, preferably at most seven, preferably at most six, preferably at most five, preferably at most four, preferably at most three, preferably at most two, preferably at most one amino acid in the amino acid sequence defined in SEQ ID NO: 10 are replaced by other naturally occurring amino acids.

[0057] In a further preferred embodiment of the invention, at least one inactivated gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is deleted.

[0058] In a preferred embodiment of the invention, at least one inactivated gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is non-functional.

[0059] In a particularly preferred embodiment, the at least one inactivated gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is inactivated by gene knockout, in particular by gene replacement. Preferably, the at least one inactivated gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is inactivated by gene replacement with a deletion cassette.

[0060] In a preferred embodiment of the invention, at least one gene encoding mannitol-1-phosphate-5-dehydrogenase of the genetically modified saprophytic fungus is inactivated, in particular made non-functional, by genome editing, in particular by meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or by the clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0061] In a particularly preferred embodiment, the genetically modified saprophytic fungus comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five further inactivated genes. Preferably, the at least one further inactivated gene of the genetically modified saprophytic fungus is selected from the group consisting of a gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), a gene encoding an erythritol utilization factor (EUF), a gene encoding erythrulose kinase (EYK1), a gene encoding erythritol dehydrogenase (EYD1), a gene encoding erythritol isomerase 1 (EYI1), and a gene encoding erythritol isomerase 2 (EYI2).

[0062] In a preferred embodiment of the invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1). Preferably, the at least one gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 11 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 11 prior to inactivation.

[0063] Preferably, at least one gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) of the genetically modified saprophytic fungus comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 11 before inactivation. In a further preferred embodiment, at least one gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) of the genetically modified saprophytic fungus consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 11 before inactivation.

[0064] Preferably, the phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) encoded by the nucleotide sequence of SEQ ID NO:11 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:12.

[0065] In a preferred embodiment, phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) encoded by the nucleotide sequence of SEQ ID NO: 11 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 12. Particularly preferably, phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) encoded by the nucleotide sequence of SEQ ID NO: 11 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 12.

[0066] Preferably, phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) encoded by the nucleotide sequence of SEQ ID NO: 11 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids are replaced by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 12. In a preferred embodiment of the present invention, phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps1) encoded by the nucleotide sequence of SEQ ID NO: 11 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid is replaced by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 12.

[0067] In another embodiment of the invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding an erythritol utilization factor (EUF). Preferably, the at least one gene encoding an erythritol utilization factor (Euf1) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 13 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 13 prior to inactivation.

[0068] Preferably, at least one gene encoding an erythritol utilization factor (Euf1) of the genetically modified saprophytic fungus comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 13 before inactivation. Particularly preferably, at least one gene encoding an erythritol utilization factor (Euf1) of the genetically modified saprophytic fungus consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 13 before inactivation.

[0069] Preferably, the erythritol utilization factor (Euf1) encoded by the nucleotide sequence of SEQ ID NO:13 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:14.

[0070] In a preferred embodiment, the erythritol utilization factor (Euf1) encoded by the nucleotide sequence of SEQ ID NO: 13 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 14. Particularly preferably, the erythritol utilization factor (Euf1) encoded by the nucleotide sequence of SEQ ID NO: 13 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 14.

[0071] Preferably, the erythritol utilization factor (Euf1) encoded by the nucleotide sequence of SEQ ID NO: 13 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 14 are replaced by other naturally occurring amino acids. In a preferred embodiment of the present invention, the erythritol utilization factor (Euf1) encoded by the nucleotide sequence of SEQ ID NO: 13 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 14 are replaced by other naturally occurring amino acids.

[0072] In a particularly preferred embodiment of the present invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding erythrulose kinase (EYK1). Preferably, the at least one gene encoding erythrulose kinase (Eyk1) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 15 before inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 15 before inactivation.

[0073] According to another preferred embodiment of the present invention, the at least one gene encoding erythrulose kinase (Eyk1) of the genetically modified saprophytic fungus comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 15 before inactivation. Particularly preferably, the at least one gene encoding erythrulose kinase (Eyk1) of the genetically modified saprophytic fungus consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 15 before inactivation.

[0074] Preferably, the erythrulose kinase (Eyk1) encoded by the nucleotide sequence of SEQ ID NO:15 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:16.

[0075] In a preferred embodiment, the erythrulose kinase (Eyk1) encoded by the nucleotide sequence of SEQ ID NO: 15 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 16. Particularly preferably, the erythrulose kinase (Eyk1) encoded by the nucleotide sequence of SEQ ID NO: 15 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 16.

[0076] Preferably, the erythrulose kinase (Eyk1) encoded by the nucleotide sequence of SEQ ID NO: 15 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids are replaced by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 16. In a preferred embodiment of the present invention, the erythrulose kinase (Eyk1) encoded by the nucleotide sequence of SEQ ID NO: 15 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid is replaced by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 16.

[0077] In a preferred embodiment of the invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding erythritol dehydrogenase (EYD1). Preferably, the at least one gene encoding erythritol dehydrogenase (Eyd1) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 17 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 17 prior to inactivation.

[0078] Preferably, at least one gene encoding erythritol dehydrogenase (Eyd1) of the genetically modified saprophytic fungus comprised a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 17 before inactivation. In another preferred embodiment of the present invention, at least one gene encoding erythritol dehydrogenase (Eyd1) of the genetically modified saprophytic fungus consisted of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 17 before inactivation.

[0079] Preferably, the erythritol dehydrogenase (Eyd1) encoded by the nucleotide sequence of SEQ ID NO:17 prior to inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:18.

[0080] In a preferred embodiment, the erythritol dehydrogenase (Eyd1) encoded by the nucleotide sequence of SEQ ID NO: 17 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 18. Particularly preferably, the erythritol dehydrogenase (Eyd1) encoded by the nucleotide sequence of SEQ ID NO: 17 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 18.

[0081] Preferably, the erythritol dehydrogenase (Eyd1) encoded by the nucleotide sequence of SEQ ID NO: 17 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 18 are replaced by other naturally occurring amino acids. In a preferred embodiment of the present invention, the erythritol dehydrogenase (Eyd1) encoded by the nucleotide sequence of SEQ ID NO: 17 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 18 are replaced by other naturally occurring amino acids.

[0082] In a preferred embodiment of the invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding erythritol isomerase 1 (EYI1). Preferably, the at least one gene encoding erythritol isomerase 1 (EYI1) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 19 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 19 prior to inactivation.

[0083] Particularly preferably, the at least one gene encoding erythritol isomerase 1 (Eyi1) of the genetically modified saprophytic fungus comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 19 before inactivation. In a further preferred embodiment of the present invention, the at least one gene encoding erythritol isomerase 1 (Eyi1) of the genetically modified saprophytic fungus consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 19 before inactivation.

[0084] Preferably, the erythritol isomerase 1 (Eyi1) encoded by the nucleotide sequence of SEQ ID NO: 19 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO: 20.

[0085] In a preferred embodiment, the erythritol isomerase 1 (Eyi1) encoded by the nucleotide sequence of SEQ ID NO: 19 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 20. Particularly preferably, the erythritol isomerase 1 (Eyi1) encoded by the nucleotide sequence of SEQ ID NO: 19 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 20.

[0086] Preferably, the erythritol isomerase 1 (Eyi1) encoded by the nucleotide sequence of SEQ ID NO: 19 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids are substituted by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 20. In a preferred embodiment of the present invention, the erythritol isomerase 1 (Eyi1) encoded by the nucleotide sequence of SEQ ID NO: 19 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid is substituted by other naturally occurring amino acids in the amino acid sequence defined in SEQ ID NO: 20.

[0087] In another preferred embodiment of the invention, the at least one further inactivated gene of the genetically modified saprophytic fungus is a gene encoding erythritol isomerase 2 (EYI2). Preferably, the at least one gene encoding erythritol isomerase 2 (Eyi2) of the genetically modified saprophytic fungus comprises the nucleotide sequence of SEQ ID NO: 21 prior to inactivation, in particular consists of the nucleotide sequence of SEQ ID NO: 21 prior to inactivation.

[0088] Preferably, at least one gene encoding erythritol isomerase 2 (Eyi2) of the genetically modified saprophytic fungus comprised a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 21 before inactivation. In a preferred embodiment of the present invention, at least one gene encoding erythritol isomerase 2 (Eyi2) of the genetically modified saprophytic fungus consisted of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 21 before inactivation.

[0089] Preferably, the erythritol isomerase 2 (Eyi2) encoded by the nucleotide sequence of SEQ ID NO:21 before inactivation comprises, in particular consists of, the amino acid sequence of SEQ ID NO:22.

[0090] In a preferred embodiment, the erythritol isomerase 2 (Eyi2) encoded by the nucleotide sequence of SEQ ID NO: 21 before inactivation comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 22. Particularly preferably, the erythritol isomerase 2 (Eyi2) encoded by the nucleotide sequence of SEQ ID NO: 21 before inactivation consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 22.

[0091] Preferably, the erythritol isomerase 2 (Eyi2) encoded by the nucleotide sequence of SEQ ID NO: 21 before inactivation comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 22 are replaced by other naturally occurring amino acids. In a preferred embodiment of the present invention, the erythritol isomerase 2 (Eyi2) encoded by the nucleotide sequence of SEQ ID NO: 21 before inactivation comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 22 are replaced by other naturally occurring amino acids.

[0092] In a further preferred embodiment of the invention, at least one further inactivated gene selected from the group consisting of the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), the gene encoding erythritol utilization factor (EUF), the gene encoding erythrulose kinase (EYK1), the gene encoding erythritol dehydrogenase (EYD1), the gene encoding erythritol isomerase (EYI1), and the gene encoding erythritol isomerase (EYI2) of the genetically modified saprophytic fungus is deleted.

[0093] Preferably, at least one further inactivated gene selected from the group consisting of the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), the gene encoding an erythritol utilization factor (EUF), the gene encoding erythrulose kinase (EYK1), the gene encoding erythritol dehydrogenase (EYD1), the gene encoding erythritol isomerase (EYI1), and the gene encoding erythritol isomerase (EYI2) of the genetically modified saprophytic fungus is non-functional.

[0094] In a particularly preferred embodiment, the at least one further inactivated gene selected from the group consisting of the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), the gene encoding an erythritol utilization factor (EUF), the gene encoding erythrulose kinase (EYK1), the gene encoding erythritol dehydrogenase (EYD1), the gene encoding erythritol isomerase (EYI1) and the gene encoding erythritol isomerase (EYI2) of the genetically modified saprophytic fungus is inactivated by gene knockout, in particular by gene replacement. Preferably, at least one further inactivated gene selected from the group consisting of the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), the gene encoding erythritol utilization factor (EUF), the gene encoding erythrulose kinase (EYK1), the gene encoding erythritol dehydrogenase (EYD1), the gene encoding erythritol isomerase (EYI1), and the gene encoding erythritol isomerase (EYI2) of the genetically modified saprophytic fungus is inactivated by gene replacement with a deletion cassette.

[0095] In a preferred embodiment of the invention, at least one further inactivated gene selected from the group consisting of the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase 1 (Dhaps-1), the gene encoding erythritol utilization factor (EUF), the gene encoding erythrulose kinase (EYK1), the gene encoding erythritol dehydrogenase (EYD1), the gene encoding erythritol isomerase (EYI1) and the gene encoding erythritol isomerase (EYI2) of the genetically modified saprophytic fungus is inactivated, in particular rendered non-functional, by genome editing, in particular by meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or the clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0096] In a further preferred embodiment of the present invention, the genetically modified saprophytic fungus further comprises at least one gene encoding at least one transketolase. Preferably, the at least one gene encoding at least one transketolase is tkl1, in particular a codon-optimized tkl1. Preferably, the genetically modified saprophytic fungus further comprises at least one gene encoding tkl1 from T. reesei, in particular a codon-optimized tkl1 from T. reesei. In a preferred embodiment, the at least one gene encoding at least one transketolase comprises the nucleotide sequence of SEQ ID NO:5, in particular consists of the nucleotide sequence of SEQ ID NO:5.

[0097] In a further preferred embodiment of the invention, the at least one gene encoding the at least one transketolase comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 5. Preferably, the at least one gene encoding the at least one transketolase consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 5.

[0098] Preferably, the at least one transketolase comprises, in particular consists of, the amino acid sequence of SEQ ID NO:6.

[0099] In another preferred embodiment of the invention, the at least one transketolase comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 6. Particularly preferred, the at least one transketolase consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 6.

[0100] In a further preferred embodiment of the invention, at least one transketolase comprises a sequence in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids in the amino acid sequence defined in SEQ ID NO: 6 are replaced by other naturally occurring amino acids. In a particularly preferred embodiment, at least one transketolase comprises a sequence in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid in the amino acid sequence defined in SEQ ID NO: 6 are replaced by other naturally occurring amino acids.

[0101] In a preferred embodiment of the present invention, the genetically modified saprophytic fungus further comprises at least one gene encoding at least one transaldolase. Preferably, the at least one gene encoding at least one transaldolase is tal1, in particular a codon-optimized tal1. Preferably, the genetically modified saprophytic fungus further comprises at least one gene encoding tal1 from T. reesei, in particular a codon-optimized tal1 from T. reesei. In a preferred embodiment, the at least one gene encoding at least one transaldolase comprises the nucleotide sequence of SEQ ID NO: 7, in particular consists of the nucleotide sequence of SEQ ID NO: 7.

[0102] In another preferred embodiment, the at least one gene encoding the at least one transaldolase comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 7. Preferably, the at least one gene encoding the at least one transaldolase consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 7.

[0103] Preferably, the at least one transaldolase comprises, in particular consists of, the amino acid sequence of SEQ ID NO:8.

[0104] Particularly preferably, the at least one transaldolase comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 8. Preferably, the at least one transaldolase consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 8.

[0105] In another preferred embodiment of the invention, the at least one transaldolase comprises an amino acid sequence as defined in SEQ ID NO: 8, in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids are substituted by other naturally occurring amino acids. In a particularly preferred embodiment, the at least one transaldolase comprises an amino acid sequence as defined in SEQ ID NO: 8, in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid is substituted by other naturally occurring amino acids.

[0106] In a further preferred embodiment of the present invention, the genetically modified saprophytic fungus further comprises at least one gene encoding at least one erythritol utilisation factor (EUF). Preferably, the at least one gene encoding at least one erythritol utilisation factor is euf1, in particular a codon-optimised euf1. Preferably, the genetically modified saprophytic fungus further comprises at least one gene encoding euf1 from T. reesei, in particular a codon-optimised euf1 from T. reesei. In a preferred embodiment, the at least one gene encoding at least one erythritol utilisation factor (EUF) comprises, in particular consists of, the nucleotide sequence of SEQ ID NO: 13.

[0107] In a preferred embodiment, the at least one gene encoding the at least one erythritol utilization factor of the genetically modified saprophytic fungus comprises a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 13. Preferably, the at least one gene encoding the at least one erythritol utilization factor consists of a nucleotide sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 13.

[0108] Preferably, at least one erythritol utilisation factor (EUF) comprises, in particular consists of, the amino acid sequence of SEQ ID NO:14.

[0109] In a further preferred embodiment, the at least one erythritol utilisation factor comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 14. Preferably, the at least one erythritol utilisation factor consists of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to SEQ ID NO: 14.

[0110] In a further preferred embodiment of the invention, the at least one erythritol utilization factor comprises an amino acid sequence as defined in SEQ ID NO: 14 in which 1, preferably 2, preferably 3, preferably 5, preferably 6, preferably 7, preferably 8, preferably 9, preferably 10 amino acids are substituted by other naturally occurring amino acids. In a particularly preferred embodiment, the at least one erythritol utilization factor comprises an amino acid sequence as defined in SEQ ID NO: 14 in which at most 10, preferably at most 9, preferably at most 8, preferably at most 7, preferably at most 6, preferably at most 5, preferably at most 4, preferably at most 3, preferably at most 2, preferably at most 1 amino acid is substituted by other naturally occurring amino acids.

[0111] In a preferred embodiment of the invention, at least one gene encoding at least one membrane-bound alditol transporter, at least one gene encoding at least one erythrose reductase, at least one gene encoding at least one transketolase, at least one gene encoding at least one transaldolase, and / or at least one gene encoding at least one erythritol utilization factor (EUF) of the genetically modified saprophytic fungus are foreign polynucleotide sequences.

[0112] Preferably, at least one gene encoding at least one membrane-bound alditol transporter, at least one gene encoding at least one erythrose reductase, at least one gene encoding at least one transketolase, at least one gene encoding at least one transaldolase, and / or at least one gene encoding at least one erythritol utilization factor (EUF) of the genetically modified saprophytic fungus are endogenous polynucleotide sequences.

[0113] In a further preferred embodiment, the genetically modified saprophytic fungus comprises at least one exogenous gene encoding at least one membrane-bound alditol transporter. Preferably, the genetically modified saprophytic fungus comprises at least one endogenous gene encoding at least one membrane-bound alditol transporter.

[0114] Preferably, the genetically modified saprophytic fungus comprises at least one foreign gene encoding at least one erythrose reductase. In another preferred embodiment, the genetically modified saprophytic fungus comprises at least one endogenous gene encoding at least one erythrose reductase.

[0115] More preferably, the genetically modified saprophytic fungus comprises at least one exogenous gene encoding at least one transketolase. In a preferred embodiment of the present invention, the genetically modified saprophytic fungus comprises at least one endogenous gene encoding at least one transketolase.

[0116] Preferably, the genetically modified saprophytic fungus comprises at least one foreign gene encoding at least one transaldolase. Preferably, the genetically modified saprophytic fungus comprises at least one endogenous gene encoding at least one transaldolase.

[0117] Preferably, the genetically modified saprophytic fungus comprises at least one exogenous gene encoding at least one erythritol utilisation factor (EUF). Preferably, the genetically modified saprophytic fungus comprises at least one endogenous gene encoding at least one erythritol utilisation factor (EUF).

[0118] In a preferred embodiment of the invention, at least one gene encoding at least one membrane-bound alditol transporter, at least one gene encoding at least one erythrose reductase, at least one gene encoding at least one transketolase, at least one gene encoding at least one transaldolase and / or at least one gene encoding at least one erythritol utilization factor (EUF) are stably or transiently introduced into the genome of the genetically modified saprophytic fungus.

[0119] In a particularly preferred embodiment of the invention, at least one gene encoding at least one membrane-bound alditol transporter, at least one gene encoding at least one erythrose reductase, at least one gene encoding at least one transketolase, at least one gene encoding at least one transaldolase and / or at least one gene encoding at least one erythritol utilization factor (EUF) are overexpressed.

[0120] Preferably, at least one gene encoding at least one membrane-bound alditol transporter, in particular at least one foreign or endogenous gene encoding at least one membrane-bound alditol transporter, of the genetically modified saprophytic fungus is overexpressed. In a further preferred embodiment, at least one gene encoding at least one erythrose reductase, in particular at least one foreign or endogenous gene encoding at least one erythrose reductase, of the genetically modified saprophytic fungus is overexpressed. Preferably, at least one gene encoding at least one transketolase, in particular at least one foreign or endogenous gene encoding at least one transketolase, of the genetically modified saprophytic fungus is overexpressed. Further preferably, at least one gene encoding at least one transaldolase, in particular at least one foreign or endogenous gene encoding at least one transaldolase, of the genetically modified saprophytic fungus is overexpressed. Preferably, at least one gene encoding at least one erythritol utilisation factor (EUF), in particular at least one foreign or endogenous gene encoding at least one erythritol utilisation factor (EUF), of the genetically modified saprophytic fungus is overexpressed.

[0121] In a further preferred embodiment of the present invention, 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, the at least one gene encoding at least one transaldolase and / or the at least one gene encoding at least one erythritol utilization factor (EUF) of the genetically modified saprophytic fungus are under the control of a constitutive or inducible promoter. In a preferred embodiment, the constitutive or inducible promoter is a naturally occurring promoter. In a further preferred embodiment, the constitutive or inducible promoter is a synthetic promoter. Preferably, the constitutive promoter is selected from pki, tef, gpd. Preferably, the inducible promoter is selected from bga1, bxl1, cbh1, cbh2, xyn1, xyn2. Preferably, the at least one gene encoding at least one membrane-bound alditol transporter of the genetically modified saprophytic fungus is under the control of a promoter selected from pki, tef, gpd, preferably under the control of the pki promoter. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase is under the control of a promoter selected from pki, tef, gpd, preferably under the control of the tef promoter. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one transketolase is under the control of a promoter selected from pki, tef, gpd, preferably under the control of the tef promoter. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one transaldolase is under the control of a promoter selected from pki, tef, gpd, preferably under the control of the tef promoter. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one erythritol utilization factor (EUF) is under the control of a promoter selected from pki, tef, gpd, preferably under the control of the tef promoter.

[0122] In a further preferred embodiment of the present invention, at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter, at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase, at least one gene of the genetically modified saprophytic fungus encoding at least one transketolase, at least one gene of the genetically modified saprophytic fungus encoding at least one transaldolase, and / or at least one gene of the genetically modified saprophytic fungus encoding at least one erythritol utilization factor (EUF) are present on a plasmid. Preferably, at least one gene of the genetically modified saprophytic fungus encoding at least one membrane-bound alditol transporter, at least one gene of the genetically modified saprophytic fungus encoding at least one erythrose reductase, at least one gene of the genetically modified saprophytic fungus encoding at least one transketolase, at least one gene of the genetically modified saprophytic fungus encoding at least one transaldolase, and / or at least one gene of the genetically modified saprophytic fungus encoding at least one erythritol utilization factor (EUF) are each present on a separate plasmid.

[0123] In a particularly preferred embodiment of the invention, the genetically modified saprophytic fungus is the Trichoderma reesei strain deposited at the Westerdijk Fungal Biodiversity Institute under CBS number 146708.

[0124] According to a preferred embodiment of the present invention, the at least one saprophytic fungus is cultivated in step a) in a volume of at least 5 L, preferably at least 10 L, preferably at least 25 L, preferably at least 50 L, preferably at least 100 L, preferably at least 250 L, preferably at least 500 L, preferably at least 1,000 L, preferably at least 2,500 L, preferably at least 7,500 L, preferably at least 10,000 L, preferably at least 25,000 L, preferably at least 50,000 L, preferably at least 75,000 L, preferably at least 100,000 L.

[0125] In a further preferred embodiment of the present invention the at least one saprophytic fungus is cultivated in step a) in the medium until the medium contains erythritol at a concentration of at least 250 mg / L, preferably at least 300 mg / L, preferably at least 350 mg / L, preferably at least 400 mg / L, preferably at least 450 mg / L, preferably at least 500 mg / L, preferably at least 600 mg / L, preferably at least 700 mg / L, preferably at least 800 mg / L, preferably at least 900 mg / L, preferably at least 1 g / L, preferably at least 2 g / L, preferably at least 3 g / L, preferably at least 4 g / L, preferably at least 5 g / L, preferably at least 6 g / L, preferably at least 7 g / L, preferably at least 8 g / L, preferably at least 9 g / L, preferably at least 10 g / L, preferably at least 15 g / L, preferably at least 20 g / L, preferably at least 25 g / L, preferably at least 30 g / L, preferably at least 35 g / L, preferably at least 40 g / L.

[0126] Particularly preferably, the at least one saprophytic fungus is cultivated in the medium until the medium contains erythritol at a concentration of 250 mg / L to 40 g / L, preferably 500 mg / L to 40 g / L, preferably 750 mg / L to 40 g / L, preferably 1 g / L to 40 g / L.

[0127] In a further preferred embodiment, the at least one saprophytic fungus is cultured in the medium until the medium contains erythritol at a concentration of 5 g / L to 40 g / L, preferably 10 g / L to 40 g / L, preferably 15 g / L to 40 g / L, preferably 20 g / L to 40 g / L, preferably 25 g / L to 40 g / L, preferably 30 g / L to 40 g / L.

[0128] According to a preferred embodiment of the invention, the yield of erythritol is at least 3 mg / g, preferably at least 4 mg / g, preferably at least 5 mg / g, preferably at least 6 mg / g, preferably at least 7 mg / g, preferably at least 8 mg / g, preferably at least 9 mg / g, preferably at least 10 mg / g, preferably at least 11 mg / g, preferably at least 12 mg / g, preferably at least 13 mg / g, preferably at least 14 mg / g, preferably at least 15 mg / g, preferably at least 17.5 mg / g, preferably at least 20 mg / g, preferably at least 25 mg / g, preferably at least 30 mg / g, preferably at least 40 mg / g, preferably at least 50 mg / g, preferably at least 75 mg / g, preferably at least 100 mg / g (respectively based on mg erythritol per g carbon source consumed, in particular mg erythritol per g glucose consumed).

[0129] In a preferred embodiment, the yield of erythritol is 3-100 mg / g, preferably 4-100 mg / g, preferably 5-100 mg / g, preferably 6-100 mg / g, preferably 7-75 mg / g, preferably 8-75 mg / g, preferably 9-75 mg / g, preferably 10-75 mg / g, preferably 15-50 mg / g (based on mg of erythritol per g of carbon source consumed, in particular mg of erythritol per g of glucose consumed, respectively).

[0130] In a particularly preferred embodiment of the present invention, the at least one saprophytic fungus is cultivated in step a) in a medium with a pH in the range of 2 to 7, preferably 2.5 to 6.5, preferably 2.5 to 6, preferably 3 to 5.5. Particularly preferably, the at least one saprophytic fungus is cultivated in a medium with a pH of at most 7, preferably at most 6.5, preferably at most 6, preferably at most 5.5, preferably at most 5.

[0131] According to a preferred embodiment of the present invention, the at least one saprophytic fungus is cultivated in step a) in a medium at a temperature of 25 to 35°C, preferably 26 to 34°C, preferably 27 to 33°C, preferably 28 to 32°C, preferably 29 to 31°C, preferably 30°C.

[0132] In a preferred embodiment of the present invention, the at least one saprophytic fungus is cultivated in step a) in a medium at a temperature of at least 15°C, preferably at least 20°C, preferably at least 25°C, preferably at least 26°C, preferably at least 27°C, preferably at least 28°C.

[0133] In a further preferred embodiment, the at least one saprophytic fungus is cultivated in step a) in a culture medium at a temperature of at most 35°C, preferably at most 34°C, preferably at most 33°C, preferably at most 32°C, preferably at most 31°C.

[0134] Preferably, the at least one saprophytic fungus is cultured in step a) in the medium for at least 24 hours, preferably at least 36 hours, preferably at least 48 hours, preferably at least 60 hours, preferably at least 72 hours, preferably at least 84 hours, preferably at least 96 hours, preferably at least 108 hours, preferably at least 120 hours, preferably at least 132 hours, preferably at least 144 hours, preferably at least 156 hours, preferably at least 168 hours.

[0135] In a further preferred embodiment of the invention the at least one saprophytic fungus is cultured in step a) in the culture medium for at most 240 hours, preferably at most 216 hours, preferably at most 192 hours, preferably at most 180 hours, preferably at most 168 hours, preferably at most 156 hours, preferably at most 144 hours, preferably at most 132 hours, preferably at most 120 hours, preferably at most 108 hours, preferably at most 96 hours, preferably at most 84 hours, preferably at most 72 hours.

[0136] According to a preferred embodiment of the present invention, the cultivation of the at least one saprophytic fungus in step a) is carried out without the addition of any osmotically effective agent during the cultivation, in particular without the addition of at least one salt during the cultivation. Particularly preferably, the cultivation of the at least one saprophytic fungus in step a) is carried out without the addition of sodium chloride (NaCl) during the cultivation.

[0137] In a preferred embodiment of the present invention, the cultivation of at least one saprophytic fungus in step a) is carried out in batch mode. Preferably, the cultivation of at least one saprophytic fungus in step a) is carried out in fed-batch mode. In another preferred embodiment, the cultivation of at least one saprophytic fungus in step a) is carried out in continuous mode.

[0138] In a further preferred embodiment of the invention, erythritol is recovered in step c) by crystallization.

[0139] In a particularly preferred embodiment of the present invention, the recovery of erythritol in step b) comprises: i) removal of biomass, preferably by centrifugation and membrane filtration; ii) decolorization of the medium, preferably with activated charcoal; iii) desalting the medium, preferably desalting and decolorizing the medium by electrodialysis; iv) preparative chromatography, preferably reversed-phase chromatography using ion-exclusion and size-exclusion mechanisms; and v) Concentration and crystallization of erythritol The process includes the steps of:

[0140] According to a preferred embodiment of the present invention, a heat denaturation step is performed before, during or after step i). Preferably, a heat denaturation step is performed before step i). In a further preferred embodiment, a heat denaturation step is performed during step i). Preferably, a heat denaturation step is performed after step i).

[0141] Particularly preferably, the heat denaturation step comprises heating the medium to a temperature of at least 45°C, preferably at least 50°C, preferably at least 55°C, preferably at least 60°C, preferably at least 65°C, preferably at least 70°C, preferably 75°C, preferably at least 80°C, preferably at least 85°C, preferably at least 90°C, preferably at least 95°C.

[0142] In a preferred embodiment of the invention the medium is subjected to heat denaturation for at least 10 seconds, preferably at least 20 seconds, preferably at least 30 seconds, preferably at least 40 seconds, preferably at least 50 seconds, preferably at least 1 minute, preferably at least 2 minutes, preferably at least 3 minutes, preferably at least 4 minutes, preferably at least 5 minutes, preferably at least 6 minutes, preferably at least 7 minutes, preferably at least 8 minutes, preferably at least 9 minutes, preferably at least 10 minutes.

[0143] The heat denaturation step primarily serves to heat inactivate proteins and enzymes in the medium before further recovery steps are performed.

[0144] In a particularly preferred embodiment of the present invention, the erythritol recovered in step b), in particular the erythritol obtained in step v), has a purity of at least 90%, preferably at least 92%, preferably at least 94%, preferably at least 96%, preferably at least 97%, preferably at least 97.5%, preferably at least 98%, preferably at least 98.5%, preferably at least 99%, preferably at least 99.5%.

[0145] As used herein, the term "genetically modified" and its grammatical equivalents refer to one or more modifications of a nucleic acid (e.g., a nucleic acid in the genome of an organism, particularly a saprophytic fungus). A "genetically modified" saprophytic fungus can refer to a saprophytic fungus that has had additions, deletions and / or modifications in genes (particularly inactivated).

[0146] In the context of the present invention, the term "erythrose reductase" relates to any enzyme that catalyzes the reversible reduction of aldoses to alditols and has a predominant specific activity for the reduction of erythrose to erythritol. Therefore, the "erythrose reductase" according to the present invention exhibits high specificity and affinity for the substrate erythrose. In particular, the "erythrose reductase" has a higher specificity for erythrose than for any other substrate, such as glycerol.

[0147] The term "membrane-bound alditol transporter" as used herein refers to a membrane protein suitable for reversibly transporting alditol across the outer membrane, in particular for transporting alditol produced within the cell into the medium. According to the present invention, a "membrane-bound alditol transporter" is suitable for transporting erythritol produced within the cell across the membrane into the medium.

[0148] In the context of the present invention, the term "transketolase" refers to an enzyme of the pentose phosphate pathway that catalyzes the thiamine-dependent, reversible transfer of a C-2 unit from a ketose donor to an acceptor aldopentose. In particular, "transketolase" catalyzes the transfer of a C-2 unit from xylulose-5-phosphate to ribose-5-phosphate to form seduheptulose-7-phosphate and glyceraldehyde-3-phosphate.

[0149] The term "transaldolase" refers to an enzyme in the nonoxidative process of the pentose phosphate pathway that catalyzes the reversible transfer of a C-3 unit from a ketose donor to an acceptor aldose. In particular, "transketolase" catalyzes the transfer of a C-3 unit from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate to form erythrose-4-phosphate and fructose-6-phosphate.

[0150] In the context of the present invention, an "inactivated" gene is a gene that cannot be transcribed, either temporarily or permanently, or that can be transcribed but whose transcript is not accessible for gene translation. According to the present invention, inactivation of a gene includes, but is not limited to, partial or complete gene deletion, partial or complete gene replacement, gene suppression, gene insertion, and inactivation by mutation of a gene. "Inactivation" in the context of the present invention also includes any post-transcriptional gene regulation (especially by RNAi or siRNA) that inhibits the translation of a transcript into a gene product.

[0151] In the context of the present invention, a "non-functional" gene is a gene present in the genome of an organism, or in a plasmid within an organism, which gene is not transcribed, or which is transcribed but the transcript is not translated into a gene product.

[0152] In the context of the present invention, a "deleted" gene is a gene that was previously present in the genome of an organism or in a plasmid in an organism, but that has been completely deleted from the genome of the organism or from the plasmid in the organism.

[0153] In the context of the present invention, the verb "overexpress" means to artificially increase the amount and expression of a gene, including inducible overexpression of a controllable gene using an inducible promoter and constant overexpression of a gene using a constitutive promoter.

[0154] The terms "oligomeric sugars" or "oligomeric C-5 and C-6 sugars" are used herein for disaccharides, oligosaccharides, and polysaccharides composed of at least two monomers, preferably selected from C-5 and C-6 sugars.

[0155] In the context of the present invention, the term "a" encompasses the meaning of "one" or "one or more".

[0156] In the context of the present invention, the term "comprising" preferably has the meaning of "containing" or "including" and means including the elements specifically specified without excluding the presence of further elements. However, in preferred embodiments, the term "comprising" is also understood to have the meaning "consisting essentially of", and in more preferred embodiments, to have the meaning "consisting of".

[0157] As used herein, the term "and / or" is used to express disclosure of any combination of the individual elements listed in a list. By way of example only, the phrase "A, B, and / or C" can mean A only, B only, C only, (A and B), (B and C), (A and C), and (A, B and C).

[0158] Further preferred embodiments of the invention are the subject matter of the dependent claims.

[0159] The present invention is further illustrated by the following examples and the accompanying drawings. [Brief description of the drawings]

[0160] [Figure 1] FIG. 1 shows the effect of the carbon source concentration in the medium on erythritol production by Trichoderma reesei after 168 h of incubation in media containing 50 g / L, 70 g / L or 90 g / L glucose as the carbon source and 80 mM urea as the nitrogen source. [Diagram 2] Figure 2 shows the effect of the nitrogen source concentration in the medium on erythritol production by Trichoderma reesei after 168 h of incubation in a medium containing 70 g / L glucose as a carbon source and 20 mM, 60 mM or 80 mM urea as a nitrogen source. [Diagram 3] FIG. 3 shows the effect of initial carbon source concentration on erythritol production by the genetically engineered strain QM6a△tmus53 fps1(Reasl1) err1(Repyr4) △mpdh ("engineered strain") and the corresponding parental strain QM6a△tmus53 ("PS") after 24, 48, 72, 96 and 120 hours of incubation in medium containing 10 g / L, 50 g / L or 100 g / L glucose as the carbon source and 20 mM ammonium sulfate as the nitrogen source. [Figure 4] Figure 4 shows the effect of different nitrogen sources at concentrations of 20 mM or 80 mM on erythritol production by Trichoderma reesei (QM6a△tmus53) after 96 h of cultivation in medium containing 50 g / L glucose as the carbon source. [Diagram 5] Figure 5 shows the effect of nitrogen source on biomass production (dry weight) of Trichoderma reesei (QM6a△tmus53) after 96 h of cultivation in medium containing 50 g / L glucose as carbon source and 20 mM or 80 mM of each nitrogen source. [Figure 6]FIG. 6 shows the effect of urea concentration on erythritol production by Trichoderma reesei (QM6a△tmus53) after 168 h of cultivation in medium containing 70 g / L glucose and 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or 120 mM urea or in medium containing 50 g / L glucose and 80 mM urea (right bar).

[0161] (Example) 1. Biotechnological production of erythritol 1.1 Effect of carbon source concentration on erythritol production Trichoderma reesei was cultivated in 250 mL flasks each containing 100 mL of medium containing 50 g / L, 70 g / L or 90 g / L glucose as a carbon source and 80 mM urea as a nitrogen source.

[0162] After 168 hours of cultivation, erythritol concentrations were measured in the sample medium, and the average erythritol concentrations in the samples were 0.733 g / L (50 g / L glucose), 0.978 g / L (70 g / L glucose), and 0.952 g / L (90 g / L glucose) (see FIG. 1).

[0163] 1.2 Effect of nitrogen source concentration on erythritol production In a similar experiment, Trichoderma reesei was cultivated in 250 mL flasks each containing 100 mL of medium containing 70 g / L glucose as a carbon source and 20 mM, 60 mM, or 80 mM urea as a nitrogen source.

[0164] After 168 hours of cultivation, the erythritol concentrations in the samples were measured, and the average erythritol concentrations in the samples were 0.051 g / L (20 mM urea), 0.55 g / L (60 mM urea), and 0.978 g / L (80 mM urea) (see FIG. 2).

[0165] 1.3 Effect of initial glucose concentration on erythritol production by genetically modified strains and the corresponding parent strains Trichoderma reesei strain QM6a△tmus53 fps1(Reasl1) err1(Repyr4) △mpdh ("modified strain") and the corresponding parent strain QM6a△tmus53 ("PS") were cultured in 250 mL flasks each containing 100 mL of medium containing 10 g / L, 50 g / L or 100 g / L glucose as a carbon source and 20 mM ammonium sulfate as a nitrogen source.

[0166] After 24, 48, 72, 96, and 120 hours of cultivation, erythritol concentrations were measured in the sample media (see Table 1 and Figure 3). Erythritol could not be detected only in the medium containing 10 g / L glucose.

[0167] [Table 1]

[0168] 1.4 Effect of different nitrogen sources on erythritol production by Trichoderma reesei To investigate the effect of different nitrogen sources on erythritol production in Trichoderma reesei, strain QM6a△tmus53 was cultivated in 250 mL flasks containing 100 mL of medium containing 50 g / L glucose as carbon source and either 20 mM or 80 mM ammonium sulfate, sodium nitrate, or urea as nitrogen source.

[0169] After 96 hours of cultivation, erythritol concentration was measured in the medium of the samples. The average concentration of erythritol in the samples was 0.018 g / L (50 g / L glucose, 20 mM NH + ), 0.028g / L (50g / L glucose, 80mM NH4 +), 0.03 g / L (50 g / L glucose, 20 mM urea), and 0.301 g / L (50 g / L glucose, 80 mM urea). - No erythritol could be detected in the medium after 96 hours when erythritol was used (see FIG. 4).

[0170] 1.5 Effect of different nitrogen sources on the growth of Trichoderma reesei Trichoderma reesei (QM6a△tmus53) was cultivated in 250 mL flasks each containing 100 mL of medium containing 50 g / L glucose as a carbon source and either 20 mM or 80 mM ammonium sulfate, sodium nitrate, or urea as a nitrogen source.

[0171] After 96 hours of incubation, the dry weight of Trichoderma reesei was measured in the different samples. The average weight was 0.384 g (50 g / L glucose, 20 mM NH4 + ), 0.417g (50g / L glucose, 80mM NH4 + ), 0.107g (50g / L glucose, 20mM NO3 - ), 0.117g (50g / L glucose, 80mM NO3 - ), 0.557 g (50 g / L glucose, 20 mM urea), and 1.007 g (50 g / L glucose, 80 mM urea) (see FIG. 5).

[0172] 1.6 Effect of urea concentration in the medium on erythritol yield The effect of the urea concentration in the medium on the erythritol yield, which refers to the amount of glucose consumed by Trichoderma reesei (QM6a△tmus53) during cultivation, was investigated. For this purpose, Trichoderma reesei was cultivated in 250 mL flasks containing 100 mL of medium containing 70 g / L glucose as carbon source and 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM or 120 mM urea as nitrogen source. The erythritol yield was also measured for the cultivation of Trichoderma reesei in the presence of 100 mL of medium containing 50 g / L glucose as carbon source and 80 mM urea as nitrogen source.

[0173] After 168 hours of cultivation, the decrease in erythritol and glucose concentrations in the medium was measured in the different samples. The average erythritol yields (mg erythritol per g glucose consumed) were 3.03 mg / g (70 g / L glucose, 60 mM urea), 18.59 mg / g (70 g / L glucose, 70 mM urea), 15.2 mg / g (70 g / L glucose, 80 mM urea), 14.3 mg / g (70 g / L glucose, 90 mM urea), 9.92 mg / g (70 g / L glucose, 100 mM urea), 8.47 mg / g (50 g / L glucose, 110 mM urea), 5.23 mg / g (70 g / L glucose, 120 mM urea) and 15.43 mg / g (50 g / L glucose, 80 mM urea) (see FIG. 6).

Claims

1. 1. A method for producing erythritol, comprising: a) culturing at least one saprophytic fungus in a medium containing a carbon source and a nitrogen source at a concentration of at least 40 g / L to obtain erythritol in the medium; b) recovering erythritol from the medium; The method includes:

2. The method according to claim 1, wherein the medium comprises a carbon source at a concentration of 40 to 200 g / L, preferably 70 to 90 g / L.

3. 3. The method according to claim 1 or 2, wherein the culture medium comprises a nitrogen source at a concentration of at least 50 mM, preferably at least 60 mM, preferably at least 70 mM.

4. 4. The method of claim 1, wherein the medium comprises a lignocellulose-containing hydrolysate.

5. 5. The method according to claim 1, wherein the carbon source is glucose or xylose.

6. 5. The method according to claim 1, wherein the carbon source comprises a hydrolysate obtained by hydrothermal treatment of a raw material comprising cellulose, hemicellulose and / or starch.

7. 6. The method according to claim 1, wherein the nitrogen source is urea.

8. 8. The method according to any one of claims 1 to 7, wherein the at least one saprophytic fungus is a filamentous fungus from the genus Hypocrea, Trichoderma, Gibberella, Fusarium, Aspergillus or Penicillium.

9. 9. The method according to any one of claims 1 to 8, wherein the saprophytic fungus is selected from the group consisting of Hypocrea jecorina (Trichoderma reesei), red mold (Gibberella zeae), red mold of wheat (Fusarium graminearum), Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae and Penicillium chrysogenum.

10. 10. The method according to any one of claims 1 to 9, wherein the saprophytic fungus is Hypocrea jecorina (Trichoderma reesei).

11. 11. The method according to any one of claims 1 to 10, wherein in step a) the at least one saprophytic fungus is cultivated in the medium until the medium contains erythritol at a concentration of at least 250 mg / L, preferably at least 500 mg / L, preferably at least 1 g / L.

12. 12. The method according to any one of claims 1 to 11, wherein in step a) the at least one saprophytic fungus is cultivated in a medium at a pH ranging from 2 to 7, preferably from 3 to 5.

5.

13. 13. The method according to any one of claims 1 to 12, wherein erythritol is recovered in step b) by crystallization.

14. The recovery of erythritol in step b) i) removal of biomass, preferably by centrifugation and membrane filtration; ii) decolorization of the medium, preferably with activated charcoal; iii) desalting the medium, preferably desalting and decolorizing the medium by electrodialysis; iv) preparative chromatography, preferably reversed-phase chromatography using ion-exclusion and size-exclusion mechanisms; and v) Concentration and crystallization of erythritol The method according to any one of claims 1 to 13, comprising the steps of:

15. 15. The method of claim 14, wherein a heat denaturation step is carried out before, during or after step i).

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