WO2019219804A1 - Procédé de production d'un polypeptide - Google Patents

Procédé de production d'un polypeptide Download PDF

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Publication number
WO2019219804A1
WO2019219804A1 PCT/EP2019/062608 EP2019062608W WO2019219804A1 WO 2019219804 A1 WO2019219804 A1 WO 2019219804A1 EP 2019062608 W EP2019062608 W EP 2019062608W WO 2019219804 A1 WO2019219804 A1 WO 2019219804A1
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Prior art keywords
enzyme
sucrose
fermentation
fungus
acid
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PCT/EP2019/062608
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English (en)
Inventor
Rolf POLDERMANS
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Dsm Ip Assets B.V.
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Priority to BR112020023198-3A priority Critical patent/BR112020023198A2/pt
Publication of WO2019219804A1 publication Critical patent/WO2019219804A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis

Definitions

  • the invention relates a process for producing a polypeptide by culturing a microorganism on sucrose-containing biomass.
  • yeasts and fungi have become attractive options for expressing cellulolytic and hemicellulolytic enzymes, as they can be easily grown at a large scale in simple media, which allows low production costs.
  • filamentous fungi such as Aspergillus and Trichoderma have been developed into expression platforms for screening and production. Their ability to express native and heterologous enzymes to high levels, makes them well-suited for the large-scale production of enzymes.
  • An object of the invention is to provide an improved production process for enzymes such as cellulolytic and/or hemicellulolytic enzymes.
  • the object of the invention is to provide an improved production process for enzymes by fungi. Optimization and improvement lies in culturing a fungus using a sucrose-containing biomass, more in particular using a treated sucrose-containing biomass.
  • Described herein is a process for producing a cellulolytic and/or hemicellulolytic enzyme by culturing a filamentous fungus under conditions which allow for expression of the enzyme, wherein the process comprises the steps of (i) treating a sucrose-containing biomass at a temperature from 50°C to 180°C and a pH from 1 to 5 for 0.5 to 90 minutes to produce a treated biomass, (ii) culturing the filamentous fungus using the treated biomass as substrate under conditions conducive for production of the enzyme, and (iii) optionally, recovering the enzyme.
  • “treating a sucrose-containing biomass” can be interpreted as "incubating a sucrose-containing biomass”.
  • sucrose-containing biomass is treated at a temperature from 50°C to 180°C, from 55°C to 175°C, from 60°C to 170°C, from 65°C to 165°C, from 70°C to 160°C, from 75°C to 155°C, from 80°C to 150°C, from 85°C to 145°C, from 90°C to 140°C.
  • sucrose-containing biomass is treated at a pH from 1 to 5, a pH from 1 .5 to 4.5, a pH from 2.0 to 4.0, a pH from 2.5 to 3.5.
  • sucrose-containing biomass is treated for 0.5 to 90 minutes, for 1 to 75 minutes, for 2 to 60 minutes, for 3 to 45 minutes, for 4 to 40 minutes, for 5 to 35 minutes, for 6 to 30 minutes. Any of the above embodiments can be combined in the processes as described herein.
  • sucrose-containing biomass is treated with an acid.
  • sucrose-containing biomass is treated with an acid in combination with any of the above embodiments.
  • the acid is selected from the group consisting of phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid and citric acid.
  • sucrose-containing biomass comprises from 10% - 80% sucrose by dry weight, comprises from 20% - 70% sucrose by dry weight, comprises from 30% - 60% sucrose by dry weight, comprises from 40% - 50% sucrose by dry weight.
  • sucrose- containing biomass is selected from the group consisting of sugar cane, sugar beet, cane molasses, beet molasses and sweet sorghum.
  • the filamentous fungus is cultured using the treated biomass as substrate under conditions conducive for production of the enzyme.
  • Conducive for production of the enzyme means a suitable growth and production medium comprising carbon and nitrogen sources and inorganic salts, a suitable growth and production temperature in the range of 20°C to 60°C, a suitable growth and production pH in the range of 3 to 6, a culturing time of 3 weeks or less.
  • the process for producing a cellulolytic and/or hemicellulolytic enzyme as described herein can be preceded by a process for propagating thefilamentous fungus. Propagation may comprise several steps in shake flasks, small containers and large containers.
  • the container(s) used in the process for producing a cellulolytic and/or hemicellulolytic enzyme as described herein have a volume of at least 1 m 3 .
  • the containers have a volume of at least 1 m 3 , at least 2 m 3 , at least 3 m 3 , at least 4 m 3 , at least 5 m 3 , at least 6 m 3 , at least 7 m 3 , at least 8 m 3 , at least 9 m 3 , at least 10 m 3 , at least 15 m 3 , at least 20 m 3 , at least 25 m 3 , at least 30 m 3 , at least 35 m 3 , at least 40 m 3 , at least 45 m 3 , at least 50 m 3 , at least 60 m 3 , at least 70 m 3 , at least 75 m 3 , at least 80 m 3 , at least 90 m 3 .
  • the container(s) will be smaller than
  • the filamentous fungus is cultured in a fed-batch culture, a batch culture, a continuous culture or any combination thereof.
  • the fungus is cultured in a fed-batch culture.
  • a person skilled in the art is well aware of the various modes of culturing and its conditions.
  • the culturing is conducted under aerobic conditions.
  • a person skilled in the art is well aware of fermentor designs for aerobic cultivation such as for instance stirred tanks and bubble columns.
  • the enzyme is recovered. Recovery may take place during and/or after expression of the enzyme.
  • Methods for recovering enzymes from the fungus, the culture medium or both are known to the skilled artisan and include, but are not limited to, biomass removal, ultrafiltration and chromatography.
  • the enzyme is not recovered and is part of a whole fermentation broth.
  • the enzyme is in the form of a whole fermentation broth of a fungus.
  • a whole fermentation broth can be prepared by culturing non-recombinant and/or recombinant fungi.
  • the fungus is a recombinant fungus comprising one or more genes which can be homologous or heterologous to the fungus.
  • the fungus is a recombinant fungus comprising one or more genes which can be homologous or heterologous to the fungus wherein the one or more genes encode enzymes that can degrade a cellulosic substrate.
  • the fungus is a non-recombinant fungus comprising one or more genes which are homologous to the fungus.
  • the fungus is a non-recombinant fungus comprising one or more genes which are homologous to the fungus wherein the one or more genes encode enzymes that can degrade a cellulosic substrate.
  • the whole fermentation broth may comprise any of the enzymes or any combination thereof.
  • the cells are killed in the whole fermentation broth.
  • the whole fermentation broth may contain organic acid(s) (used for killing the cells), killed cells and/or cell debris, and culture medium.
  • the fungus may be altered to improve or to make the enzymes.
  • the fungus may be mutated by classical strain improvement procedures or by recombinant DNA techniques. Therefore, the fungi mentioned herein can be used as such to produce the enzymes or may be altered to increase the production or to produce altered enzymes which might include heterologous enzymes, e.g. cellulases, thus enzymes that are not originally produced by that fungus.
  • a fungus more preferably a filamentous fungus, is used to produce the enzymes.
  • the enzymes produced by the fungus according to the processes as described herein are preferably cellulolytic and/or hemicellulolytic enzymes.
  • a thermophilic or thermotolerant filamentous fungus is used.
  • an additional inducer is used that induces the expression of the enzymes by the fungus.
  • the fungi are cultivated in a cell culture medium suitable for production of the enzyme of interest.
  • the enzyme may be an enzyme capable of hydrolyzing a cellulosic substrate. Suitable culture media are known in the art.
  • the whole fermentation broth can be prepared by growing the fungi to stationary phase and maintaining the fungi under limiting carbon conditions for a period of time sufficient to express the enzyme. Once the enzyme of interest is secreted by the fungi into the fermentation medium, the whole fermentation broth can be used.
  • the whole fermentation broth may comprise fungi.
  • the whole fermentation broth comprises the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the whole fermentation broth comprises the spent culture medium and cell debris present after the fungi is grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis.
  • the whole fermentation broth comprises the spent cell culture medium, extracellular enzymes and fungi.
  • the fungi present in whole fermentation broth can be lysed, permeabilized, or killed using methods known in the art to produce a cell-killed whole fermentation broth.
  • the whole fermentation broth is a cell-killed whole fermentation broth, wherein the whole fermentation broth containing the fungi cells are lysed or killed.
  • the cells are killed by lysing the fungi by chemical and/or pH treatment to generate the cell-killed whole broth of a fermentation of the fungi. In some embodiments, the cells are killed by lysing the fungi by chemical and/or pH treatment and adjusting the pH of the cell-killed fermentation mix to a suitable pH.
  • the whole fermentation broth comprises a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or any combination thereof and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or any combination thereof.
  • whole fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • whole fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the whole fermentation broth is unfractionated and comprises spent cell culture medium, extracellular polypeptides, and microbial, preferably non- viable, cells.
  • the whole fermentation broth can be fractionated and the one or more of the fractionated contents can be used.
  • the killed cells and/or cell debris can be removed from a whole fermentation broth to provide a composition that is free of these components.
  • the whole fermentation broth may further comprise a preservative and/or anti-microbial agent.
  • a preservative and/or anti-microbial agent are known in the art.
  • the whole fermentation broth as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified whole fermentation broth.
  • the whole fermentation broth may be supplemented with one or more enzymes.
  • the whole fermentation broth may be supplemented with one or more enzyme activities that are not expressed endogenously, or expressed at relatively low level by the fungi, to improve the degradation of the cellulosic substrate, for example, to fermentable sugars such as glucose or xylose.
  • the supplemental enzyme(s) can be added as a supplement to the whole fermentation broth and the enzyme(s) may be a component of a separate whole fermentation broth, or may be purified, or minimally recovered and/or purified.
  • the whole fermentation broth may be supplemented with at least another whole fermentation broth.
  • the other whole fermentation broth may be derived from the same type of fungus or from another type of fungus.
  • the whole fermentation broth comprises a whole fermentation broth of a fermentation of a recombinant fungus overexpressing one or more enzymes.
  • the whole fermentation broth comprises a whole fermentation broth of a fermentation of a recombinant fungus overexpressing one or more enzymes to improve the degradation of the cellulosic substrate.
  • the whole fermentation broth can comprise a mixture of a whole fermentation broth of a fermentation of a non-recombinant fungus and a recombinant fungus overexpressing one or more enzymes.
  • the whole fermentation broth can comprise a mixture of a whole fermentation broth of a fermentation of a non-recombinant fungus and a recombinant fungus overexpressing one or more enzymes to improve the degradation of the cellulosic substrate.
  • the whole fermentation broth comprises a whole fermentation broth of a fermentation of a fungus overexpressing beta-glucosidase.
  • the whole fermentation broth can comprise a mixture of a whole fermentation broth of a fermentation of a non-recombinant fungus and a whole fermentation broth of a fermentation of a recombinant fungus overexpressing a beta- glucosidase.
  • the filamentous fungus is selected from the group of genera consisting of Acremonium, Agaricus, Aspergillus, Aureobasidium, Beauvaria, Cephalosporium, Ceriporiopsis, Chaetomium paecilomyces, Chrysosporium, Claviceps, Cochiobolus, Coprinus, Cryptococcus, Cyathus, Emericella, Endothia, Endothia mucor, Filibasidium, Fusarium, Geosmithia, Gilocladium, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Podospora, Pyricularia, Rasamsonia, Rhizomucor, Rhizopus, Scylatidium, Schizophyllum, Stagonospora, Talaromy
  • Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • Several strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
  • ATCC American Type Culture Collection
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • strains examples include Aspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 101 1 , ATCC 9576, ATCC14488-14491 , ATCC 1 1601 , ATCC12892, P.
  • Rasamsonia is a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species (J. Floubraken et al., vida supra). Based on phenotypic, physiological and molecular data, Floubraken et al. proposed to transfer the species Talaromyces emersonii, Talaromyces byssochlamydoides, Talaromyces eburneus, Geosmithia argillacea and Geosmithia cylindrospora to Rasamsonia gen. nov.
  • Preferred fungi are Rasamsonia byssochlamydoides, Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus, Thermoascus crustaceus, Thermoascus thermophilus and Thermoascus aurantiacus, with Rasamsonia emersonii being most preferred.
  • Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsonia emersonii are used interchangeably herein.
  • the enzyme produced by the processes as described herein has carbohydrate material degrading and/or carbohydrate hydrolysing activity.
  • the enzyme that is produced by the fungus has carbohydrate material degrading and/or carbohydrate hydrolysing activity.
  • the enzyme is a cellulolytic and/or hemicellulolytic enzyme.
  • the enzyme is native to the fungus. In another embodiment the enzyme is heterologous to the fungus.
  • the term "heterologous" as used herein refers to an enzyme that is not naturally occurring in a host cell. It may be a variant of a native enzyme, but may also be an enzyme of another species.
  • an enzyme of Rasamsonia when expressed by Aspergillus, is considered to be heterologous.
  • An enzyme of Rasamsonia emersonii when expressed by Rasamsonia byssochlamydoides, is also considered to be heterologous.
  • An enzyme of a specific Rasamsonia emersonii strain when expressed by another Rasamsonia emersonii strain is however considered to be native.
  • a synthetic gene is introduced into a strain and this gene encodes for an enzyme that is identical to the native enzyme found in the strain, the enzyme encoded by the synthetic gene is also considered to be native.
  • the fungus is overexpressing the enzyme.
  • the fungus may comprise more than one copy of a polynucleotide encoding the native or heterologous enzyme.
  • the enzyme is a cellulase, hemicellulase and/or pectinase.
  • the fungus may also produce two or more, for example, three, four, five, six, seven, eight, nine or even more enzymes. Some enzymes may be native while others are heterologous.
  • the fungus produces at least two cellulases. The at least two cellulases may contain the same or different activities.
  • the fungus may produce a cellulase and/or a hemicellulase and/or a pectinase from a source other than the fungus.
  • the produced enzyme may be combined with one or more other enzymes. The combination of enzymes can then for intstance be used in a process for producing a sugar product from carbohydrate material or in a process for producing a fermentation product from carbohydrate material as described herein.
  • the enzyme is selected from the group consisting of a cellobiohydrolase, an endoglucanase, a beta-glucosidase, a beta-xylosidase, an endoxylanase, a lytic polysaccharide monooxygenase and any combination thereof.
  • a beta-glucosidase (EC 3.2.1.21 ) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing b-D-glucose residues with release of b-D- glucose.
  • a polypeptide may have a wide specificity for b-D-glucosides and may also hydrolyze one or more of the following: a b-D-galactoside, an a-L-arabinoside, a b-D-xyloside or a b-D- fucoside.
  • This enzyme may also be referred to as amygdalase, b-D-glucoside glucohydrolase, cellobiase or gentobiase.
  • an enzyme composition as described herein comprises a beta- glucosidase from Aspergillus , such as Aspergillus oryzae , such as the one disclosed in WO 02/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as the one disclosed as SEQ ID NO:2 in WO 2005/047499 or SEQ ID NO:5 in WO 2014/130812 or an Aspergillus fumigatus beta-glucosidase variant, such as one disclosed in WO 2012/044915, such as one with the following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 5 in WO 2014/130812 for numbering), or Aspergillus aculeatus, Aspergillus niger or Aspergillus kawachi.
  • Aspergillus oryzae such as the one disclosed in WO 02/09
  • the beta-glucosidase is derived from Penicillium, such as Penicillium brasilianum disclosed as SEQ ID NO:2 in WO 2007/019442, or from Trichoderma, such as Trichoderma reesei , such as ones described in US 6,022,725, US 6,982,159, US 7,045,332, US 7,005,289, US 2006/0258554 US 2004/0102619. In an embodiment even a bacterial beta-glucosidase can be used.
  • the beta-glucosidase is derived from Thielavia terrestris (WO 201 1/035029) or Trichophaea saccata (WO 2007/019442).
  • the enzyme composition comprises a beta-glucosidase from Rasamsonia, such as Rasamsonia emersonii (see WO 2012/000886).
  • endoglucanases are enzymes which are capable of catalyzing the endohydrolysis of 1 ,4-P-D-glucosidic linkages in cellulose, lichenin or cereal b-D-glucans. They belong to EC 3.2.1 .4 and may also be capable of hydrolyzing 1 ,4-linkages in b-D-glucans also containing 1 ,3-linkages.
  • Endoglucanases may also be referred to as cellulases, avicelases, b-1 ,4- endoglucan hydrolases, b-1 ,4-glucanases, carboxymethyl cellulases, celludextrinases, endo-1 ,4- b-D-glucanases, endo-1 ,4 ⁇ -D-glucanohydrolases or endo-1 ,4 ⁇ -glucanases.
  • the endoglucanase comprises a GH5 endoglucanase and/or a GH7 endoglucanase.
  • at least one of the endoglucanases in the enzyme composition is a GH5 endoglucanase or a GH7 endoglucanase.
  • these endoglucanases can be GH5 endoglucanases, GH7 endoglucanases or a combination of GH5 endoglucanases and GH7 endoglucanases.
  • the endoglucanase comprises a GH5 endoglucanase.
  • an enzyme composition as described herein comprises an endoglucanase from Trichoderma, such as Trichoderma reesei ; from Humicola , such as a strain of Humicola insolens ; from Aspergillus, such as Aspergillus aculeatus or Aspergillus kawachir, from Erwinia, such as Erwinia carotovara ; from Fusarium , such as Fusarium oxysporum ; from Thielavia, such as Thielavia terrestris ; from Humicola, such as Humicola grisea var.
  • thermoidea or Humicola insolens from Melanocarpus, such as Melanocarpus albomyces ; from Neurospora, such as Neurospora crassa from Myceliophthora, such as Myceliophthora thermophila ; from Cladorrhinum, such as Cladorrhinum foecundissimum ; and/or from Chrysosporium, such as a strain of Chrysosporium iucknowense.
  • the endoglucanase is from Rasamsonia, such as a strain of Rasamsonia emersonii (see WO 01/70998).
  • a bacterial endoglucanase can be used including, but are not limited to, Acidothermus cellulolyticus endoglucanase (see WO 91/05039; WO 93/15186; US 5,275,944; WO 96/02551 ; US 5,536,655, WO 00/70031 , WO 05/093050); Thermobifida fusca endoglucanase III (see WO 05/093050); and Thermobifida fusca endoglucanase V (see WO 05/093050).
  • a cellobiohydrolase (EC 3.2.1 .91 ) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4 ⁇ -D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the ends of the chains.
  • This enzyme may also be referred to as cellulase 1 ,4-b- cellobiosidase, 1 ,4 ⁇ -cellobiohydrolase, 1 ,4 ⁇ -D-glucan cellobiohydrolase, avicelase, bco-1 ,4-b- ⁇ - glucanase, exocellobiohydrolase or exoglucanase.
  • an enzyme composition as described herein comprises a cellobiohydrolase I from Aspergillus, such as Aspergillus fumigatus, such as the Cel7A CBH I disclosed in SEQ ID NO:6 in WO 201 1/057140 or SEQ ID NO:6 in WO 2014/130812; from Trichoderma, such as Trichoderma reeser, from Chaetomium, such as Chaetomium thermophilum- from Talaromyces , such as Talaromyces leycettanus or from Penicillium, such as Penicillium emersonii.
  • the enzyme composition comprises a cellobiohydrolase I from Rasamsonia , such as Rasamsonia emersonii (see WO 2010/122141 ).
  • an enzyme composition as described herein comprises a cellobiohydrolase II from Aspergillus , such as Aspergillus fumigatus , such as the one in SEQ ID NO:7 in WO 2014/130812 or from Trichoderma, such as Trichoderma reesei , or from Talaromyces , such as Talaromyces leycettanus , or from Thielavia, such as Thielavia terrestris , such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • the enzyme composition comprises a cellobiohydrolase II from Rasamsonia , such as Rasamsonia emersonii (see WO 201 1/098580).
  • lytic polysaccharide monooxygenases are enzymes that have recently been classified by CAZy in family AA9 (Auxiliary Activity Family 9) or family AA10 (Auxiliary Activity Family 10).
  • AA9 lytic polysaccharide monooxygenases there exist AA9 lytic polysaccharide monooxygenases and AA10 lytic polysaccharide monooxygenases.
  • Lytic polysaccharide monooxygenases are able to open a crystalline glucan structure and enhance the action of cellulases on lignocellulose substrates. They are enzymes having cellulolytic enhancing activity. Lytic polysaccharide monooxygenases may also affect cello-oligosaccharides.
  • proteins named GH61 are lytic polysaccharide monooxygenases.
  • GH61 was originally classified as endoglucanase based on measurement of very weak endo-1 ,4-P-d- glucanase activity in one family member, but have recently been reclassified by CAZy in family AA9.
  • CBM33 family 33 carbohydrate-binding module
  • CAZy has recently reclassified CBM33 in the AA10 family.
  • the lytic polysaccharide monooxygenase comprises an AA9 lytic polysaccharide monooxygenase.
  • At least one of the lytic polysaccharide monooxygenases in the enzyme composition and/or at least one of the additional lytic polysaccharide monooxygenases is an AA9 lytic polysaccharide monooxygenase.
  • all lytic polysaccharide monooxygenases in the enzyme composition and/or all additional lytic polysaccharide monooxygenases are AA9 lytic polysaccharide monooxygenase.
  • the enzyme composition comprises a lytic polysaccharide monooxygenase from Thermoascus , such as Thermoascus aurantiacus , such as the one described in WO 2005/074656 as SEQ ID NO:2 and SEQ ID NO:1 in WO2014/130812 and in WO 2010/065830; or from Thielavia, such as Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 8 or SEQ ID NO:4 in W02014/130812 and in WO 2008/148131 , and
  • WO 2011/035027 or from Aspergillus, such as Aspergillus fumigatus, such as the one described in WO 2010/138754 as SEQ ID NO:2 or SEQ ID NO: 3 in W02014/130812; or from Penicillium, such as Penicillium emersonii, such as the one disclosed as SEQ ID NO:2 in WO 201 1/041397 or SEQ ID NO:2 in W02014/130812.
  • lytic polysaccharide monooxygenases include, but are not limited to, Trichoderma reesei (see WO 2007/089290), Myceliophthora thermophila (see WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868), Penicillium pinophilum (see WO 2011/005867), Thermoascus sp. (see WO 201 1/039319), and Thermoascus crustaceous (see WO 2011/041504).
  • cellulolytic enzymes that may be comprised in the enzyme composition are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481 , WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/0521 18, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/1 17432, WO 2007/071818, WO
  • the lytic polysaccharide monooxygenase is from Rasamsonia, e.g. Rasamsonia emersonii (see WO 2012/000892).
  • an endoxylanase (EC 3.2.1 .8) is any polypeptide which is capable of catalysing the endohydrolysis of 1 ,4-P-D-xylosidic linkages in xylans.
  • This enzyme may also be referred to as endo-1 ,4-P-xylanase or 1 ,4-P-D-xylan xylanohydrolase.
  • An alternative is EC 3.2.1 .136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze 1 ,4 xylosidic linkages in glucuronoarabinoxylans.
  • the endoxylanase comprises a GH10 xylanase. This means that at least one of the endoxylanases in the enzyme composition is a GH10 xylanase. In an embodiment all endoxylanases in the enzyme composition are GH10 xylanases.
  • an enzyme composition as described herein comprises an endoxylanase from Aspergillus aculeatus (see WO 94/21785), Aspergillus fumigatus (see WO 2006/078256), Penicillium pinophilum (see WO 201 1/041405), Penicillium sp. (see WO 2010/126772), Thielavia terrestris NRRL 8126 (see WO 2009/079210), Taiaromyces leycettanus,
  • Thermobifida fusca, or Trichophaea saccata GH10 (see WO 2011/057083).
  • the enzyme composition comprises an endoxylanase from Rasamsonia , such as Rasamsonia emersonii (see WO 02/24926).
  • beta-xylosidases are polypeptides which are capable of catalysing the hydrolysis of 1 ,4-P-D-xylans, to remove successive D-xylose residues from the non reducing termini. Beta-xylosidases may also hydrolyze xylobiose. Beta-xylosidase may also be referred to as xylan 1 ,4-P-xylosidase, 1 ,4-P-D-xylan xylohydrolase, exo-1 ,4-P-xylosidase or xylobiase.
  • the beta-xylosidase comprises a GH3 beta-xylosidase. This means that at least one of the beta-xylosidases in the enzyme composition is a GH3 beta-xylosidase. In an embodiment all beta-xylosidases in the enzyme composition are GH3 beta-xylosidases.
  • an enzyme composition as described herein comprises a beta- xylosidase from Neurospora crassa, Aspergillus fumigatus or Trichoderma reesei.
  • the enzyme composition comprises a beta-xylosidase from Rasamsonia , such as Rasamsonia emersonii (see WO 2014/1 18360).
  • the enzyme as described herein may also comprises one or more of the below mentioned enzymes.
  • a b-(1 ,3)(1 ,4)-glucanase (EC 3.2.1.73) is any polypeptide which is capable of catalysing the hydrolysis of 1 ,4-P-D-glucosidic linkages in b-D-glucans containing 1 ,3- and 1 ,4- bonds.
  • Such a polypeptide may act on lichenin and cereal b-D-glucans, but not on b-D-glucans containing only 1 ,3- or 1 ,4-bonds.
  • This enzyme may also be referred to as licheninase, 1 ,3-1 ,4-b- D-glucan 4-glucanohydrolase, b-glucanase, endo-b-1 ,3-1 ,4 glucanase, lichenase or mixed linkage b-glucanase.
  • An alternative for this type of enzyme is EC 3.2.1 .6, which is described as endo- 1 ,3(4)-beta-glucanase.
  • This type of enzyme hydrolyses 1 ,3- or 1 ,4-linkages in beta-D-glucanse when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3.
  • Alternative names include endo-1 ,3-beta-glucanase, laminarinase, 1 ,3- (1 ,3;1 ,4)-beta-D-glucan 3 (4) glucanohydrolase.
  • Substrates include laminarin, lichenin and cereal beta-D-glucans.
  • an a-L-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on a-L-arabinofuranosides, a-L-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans.
  • This enzyme may also be referred to as a-N- arabinofuranosidase, arabinofuranosidase or arabinosidase.
  • arabinofuranosidases that may be comprised in the enzyme composition include, but are not limited to, arabinofuranosidases from Aspergillus niger, Humicola insolens DSM 1800 (see WO 2006/114094 and WO 2009/073383) and M. giganteus (see WO 2006/1 14094).
  • This enzyme may also be referred to as alpha-glucuronidase or alpha- glucosiduronase.
  • These enzymes may also hydrolyse 4-O-methylated glucoronic acid, which can also be present as a substituent in xylans.
  • alpha-glucuronidases that may be comprised in the enzyme composition include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus , Aspergillus fumigatus , Aspergillus niger, Aspergillus terreus, Humicola insolens (see WO 2010/014706), Penicillium aurantiogriseum (see WO 2009/068565) and Trichoderma reesei.
  • an acetyl-xylan esterase (EC 3.1 .1 .72) is any polypeptide which is capable of catalysing the deacetylation of xylans and xylo-oligosaccharides.
  • a polypeptide may catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically, not from triacetylglycerol.
  • Such a polypeptide typically does not act on acetylated mannan or pectin.
  • acetylxylan esterases that may be comprised in the enzyme composition include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (see WO 2010/108918), Chaetomium globosum , Chaetomium gracile , Humicola insolens DSM 1800 (see WO 2009/073709), Hypocrea jecorina (see WO 2005/001036), Myceliophtera thermophila (see WO 2010/014880), Neurospora crassa, Phaeosphaeria nodorum and Thielavia terrestris NRRL 8126 (see WO 2009/042846).
  • the enzyme composition comprises an acetyl xylan esterase from Rasamsonia , such as Rasamsonia emersonii (see WO 2010/000888)
  • the saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in 'natural' substrates p-nitrophenol acetate and methyl ferulate are typically poorer substrates.
  • This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin.
  • feruloyl esterases examples include, but are not limited to, feruloyl esterases form Humicola insolens DSM 1800 (see WO 2009/076122), Neosartorya fischeri, Neurospora crassa, Penicillium aurantiogriseum (see WO 2009/127729), and Thielavia terrestris (see WO 2010/053838 and WO 2010/065448).
  • the saccharide may be, for example, an oligosaccharide or a polysaccharide.
  • This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme also falls within EC 3.1 .1 .73 so may also be referred to as a feruloyl esterase.
  • an a-galactosidase (EC 3.2.1 .22) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing a-D-galactose residues in a-D-galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing a-D-fucosides. This enzyme may also be referred to as melibiase.
  • a b-galactosidase (EC 3.2.1.23) is any polypeptide which is capable of catalysing the hydrolysis of terminal non-reducing b-D-galactose residues in b-D-galactosides. Such a polypeptide may also be capable of hydrolyzing a-L-arabinosides. This enzyme may also be referred to as exo-(1 ->4) ⁇ -D-galactanase or lactase.
  • a b-mannanase (EC 3.2.1.78) is any polypeptide which is capable of catalysing the random hydrolysis of 1 ,4 ⁇ -D-mannosidic linkages in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1 ,4 ⁇ -mannosidase or endo- 1 ,4-mannanase.
  • a b-mannosidase (EC 3.2.1 .25) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing b-D-mannose residues in b-D-mannosides. This enzyme may also be referred to as mannanase or mannase.
  • an endo-polygalacturonase (EC 3.2.1 .15) is any polypeptide which is capable of catalysing the random hydrolysis of 1 ,4-a-D-galactosiduronic linkages in pectate and other galacturonans.
  • This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-a-1 ,4-galacturonide glycanohydrolase, endogalacturonase; endo-D- galacturonase or poly(1 ,4-a-D-galacturonide) glycanohydrolase.
  • the enzyme may also be known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.
  • an endo-galactanase (EC 3.2.1 .89) is any enzyme capable of catalysing the endohydrolysis of 1 ,4 ⁇ -D-galactosidic linkages in arabinogalactans.
  • the enzyme may also be known as arabinogalactan endo-1 ,4 ⁇ -galactosidase, endo-1 ,4 ⁇ -galactanase, galactanase, arabinogalactanase or arabinogalactan 4 ⁇ -D-galactanohydrolase.
  • a pectin acetyl esterase is defined herein as any enzyme which has an acetyl esterase activity which catalyses the deacetylation of the acetyl groups at the hydroxyl groups of GalUA residues of pectin.
  • an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalysing the eliminative cleavage of (1 -*4)-a-D-galacturonan methyl ester to give oligosaccharides with 4- deoxy-6-0-methyl-a-D-galact-4-enuronosyl groups at their non-reducing ends.
  • the enzyme may also be known as pectin lyase, pectin trans-e ⁇ iminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1 ®4)-6-0-methyl- a-D-galacturonan lyase.
  • a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalysing the eliminative cleavage of (1 -»4)-a-D-galacturonan to give oligosaccharides with 4-deoxy-a-D-galact- 4-enuronosyl groups at their non-reducing ends.
  • the enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, a-1 ,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo-a-1 ,4- polygalacturonic acid lyase, polygalacturonic acid lyase, pectin frans-eliminase, polygalacturonic acid frans-eliminase or (1 ®4)-a-D-galacturonan lyase.
  • an alpha rhamnosidase (EC 3.2.1 .40) is any polypeptide which is capable of catalysing the hydrolysis of terminal non-reducing a-L-rhamnose residues in a-L-rham nosides or alternatively in rhamnogalacturonan.
  • This enzyme may also be known as a-L-rhamnosidase T, a- L-rhamnosidase N or a-L-rhamnoside rhamnohydrolase.
  • exo-galacturonase (EC 3.2.1 .82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate.
  • the enzyme may also be known as exo-poly-a-galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.
  • the enzyme may also be known as galacturan 1 ,4-a-galacturonidase, exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase or poly(1 ,4-a- D-galacturonide) galacturonohydrolase.
  • exopolygalacturonate lyase (EC 4.2.2.Q) is any polypeptide capable of catalysing eliminative cleavage of 4-(4-deoxy-a-D-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate, i.e. de-esterified pectin.
  • This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid-frans-eliminase, PATE, exo-PATE, exo-PGL or (1 ®4)-a-D-galacturonan reducing-end-disaccharide-lyase.
  • rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic acid and rhamnopyranosyl in an endo-fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1 ,2-alpha-L- rhamnoyl-(1 ,4)-alpha-galactosyluronic acid].
  • rhamnogalacturonan lyase is any polypeptide which is any polypeptide which is capable of cleaving a-L-Rhap-(1 ®4)-a-D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.
  • rhamnogalacturonan acetyl esterase is any polypeptide which catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.
  • rhamnogalacturonan galacturonohydrolase is any polypeptide which is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion.
  • xylogalacturonase is any polypeptide which acts on xylogalacturonan by cleaving the b-xylose substituted galacturonic acid backbone in an encio-manner. This enzyme may also be known as xylogalacturonan hydrolase.
  • an a-L-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on a-L-arabinofuranosides, a-L-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans.
  • This enzyme may also be referred to as a-N- arabinofuranosidase, arabinofuranosidase or arabinosidase.
  • endo-arabinanase (EC 3.2.1 .99) is any polypeptide which is capable of catalysing endohydrolysis of 1 ,5-a-arabinofuranosidic linkages in 1 ,5-arabinans.
  • the enzyme may also be known as endo-arabinase, arabinan endo-1 ,5-a-L-arabinosidase, endo-1 ,5-a-L- arabinanase, endo-a-1 ,5-arabanase; endo-arabanase or 1 ,5-a-L-arabinan 1 ,5-a-L- arabinanohydrolase.
  • proteases includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4 and are suitable for use in the processes as described herein. Some specific types of proteases include, cysteine proteases including pepsin, papain and serine proteases including chymotrypsins, carboxypeptidases and metalloendopeptidases.
  • Lipase includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phospoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.
  • Liganase includes enzymes that can hydrolyze or break down the structure of lignin polymers. Enzymes that can break down lignin include lignin peroxidases, manganese peroxidases, laccases and feruloyl esterases, and other enzymes described in the art known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydrolyzing bonds formed between hemicellulosic sugars (notably arabinose) and lignin.
  • Ligninases include but are not limited to the following group of enzymes: lignin peroxidases (EC 1 .11 .1.14), manganese peroxidases (EC 1 .1 1 .1.13), laccases (EC 1.10.3.2) and feruloyl esterases (EC 3.1 .1 .73).
  • “Hexosyltransferase” (2.4.1 -) includes enzymes which are capable of catalysing a transferase reaction, but which can also catalyze a hydrolysis reaction, for example of cellulose and/or cellulose degradation products.
  • An example of a hexosyltransferase which may be used is a b-glucanosyltransferase.
  • Such an enzyme may be able to catalyze degradation of (1 ,3)(1 ,4)glucan and/or cellulose and/or a cellulose degradation product.
  • Glucuronidase includes enzymes that catalyze the hydrolysis of a glucuronoside, for example b-glucuronoside to yield an alcohol.
  • Many glucuronidases have been characterized and may be suitable for use, for example b-glucuronidase (EC 3.2.1 .31 ), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase (3.2.1 .56), glycyrrhizinate b- glucuronidase (3.2.1 .128) or a-D-glucuronidase (EC 3.2.1 .139).
  • Expansins are implicated in loosening of the cell wall structure during plant cell growth.
  • an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain.
  • CBD Carbohydrate Binding Module Family 1 domain
  • an expansin-like protein or swollenin-like protein may comprise one or both of such domains and/or may disrupt the structure of cell walls (such as disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars.
  • a cellulose induced protein for example the polypeptide product of the cip1 or cip2 gene or similar genes (see Foreman et a/., J. Biol. Chem. 278(34), 31988-31997, 2003), a cellulose/cellulosome integrating protein, for example the polypeptide product of the cipA or cipC gene, or a scaffoldin or a scaffoldin-like protein.
  • Scaffoldins and cellulose integrating proteins are multi-functional integrating subunits which may organize cellulolytic subunits into a multi-enzyme complex. This is accomplished by the interaction of two complementary classes of domain, i.e. a cohesion domain on scaffoldin and a dockerin domain on each enzymatic unit.
  • the scaffoldin subunit also bears a cellulose-binding module (CBM) that mediates attachment of the cellulosome to its substrate.
  • a scaffoldin or cellulose integrating protein may comprise one or both of such domains.
  • a catalase means a hydrogen-peroxide: hydrogen-peroxide oxidoreductase (EC 1 .1 1 .1 .6 or EC 1 .11 .1 .21 ) that catalyzes the conversion of two hydrogen peroxides to oxygen and two waters.
  • Catalase activity can be determined by monitoring the degradation of hydrogen peroxide at 240 nm based on the following reaction: 2H 2 0 2 ® 2H 2 0 + 0 2 . The reaction is conducted in 50 mM phosphate pH 7.0 at 25°C with 10.3 mM substrate (H 2 0 2 ) and approximately 100 units of enzyme per ml.
  • One catalase activity unit can be expressed as one micromole of H 2 0 2 degraded per minute at pH 7.0 and 25°C.
  • amylase as used herein means enzymes that hydrolyze alpha-1 ,4-glucosidic linkages in starch, both in amylose and amylopectin, such as alpha-amylase (EC 3.2.1 .1 ), beta- amylase (EC 3.2.1.2), glucan 1 ,4-alpha-glucosidase (EC 3.2.1.3), glucan 1 ,4-alpha- maltotetraohydrolase (EC 3.2.1 .60), glucan 1 ,4-alpha-maltohexaosidase (EC 3.2.1 .98), glucan 1 ,4- alpha-maltotriohydrolase (EC 3.2.1.1 16) and glucan 1 ,4-alpha-maltohydrolase (EC 3.2.1 .133), and enzymes that hydrolyze alpha-1 ,6-glucosidic linkages, being the branch-points in amylopectin, such as pullulanase (EC 3.2.1
  • Enzymes produced by the fungus may comprise a member of each of the classes of enzymes mentioned above, several members of one enzyme class, or any combination of these enzymes classes or helper proteins (i.e. those proteins mentioned herein which do not have enzymatic activity per se , but do nevertheless assist in biomass degradation).
  • the enzymes described above may be provided concomitantly (i.e. in a single composition of polypeptides) or separately or sequentially.
  • a process for the preparation of a sugar product from carbohydrate material comprising the steps of (a) producing a cellulolytic and/or hemicellulolytic enzyme by culturing a filamentous fungus under conditions which allow for expression of the enzyme, wherein the process comprises the step of (i) treating a sucrose-containing biomass at a temperature from 50°C to 180°C and a pH from 1 to 5 for 0.5 to 90 minutes to produce a treated biomass, (ii) culturing the filamentous fungus using the treated biomass as substrate under conditions conducive for production of the enzyme, and (iii) optionally, recovering the enzyme, (b) enzymatically hydrolysing the carbohydrate material with the enzyme to obtain the sugar product, and (c) optionally, recovering the sugar product.
  • enzymes are used, i.e. several enzymes with different cellulolytic activities are used.
  • These enzymes can be any of the enzymes described above or any combination thereof. They can be either produced by the enzyme production process as described herein.
  • the fungus can produce only one of these enzymes, but also more than one, i.e. two, three, four or even more enzymes. If not all of the enzymes necessary for the enzymatric hydrolysis are produced by the fungus, the remaining enzymes can be added after culturing. They may also be added to the carbohydrate material during enzymatic hydrolysis.
  • the carbohydrate material is cellulosic material, in particular lignocellulosic material.
  • the carbohydrate material is subjected to at least one solid/liquid separation before or during the enzymatic hydrolysis.
  • the carbohydrate material is subjected to pretreatment and at least one solid/liquid separation before or during the enzymatic hydrolysis.
  • the methods and conditions of solid/liquid separation will depend on the type of carbohydrate material used and are well within the scope of the skilled artisan. Examples include, but are not limited to, centrifugation, cyclonic separation, filtration, decantation, sieving and sedimentation. During solid/liquid separation, means and/or aids for improving the separation may be used.
  • the product of the liquefaction step can be used in the culturing of the fungus. This can be done with orwithout addition of enzymatically hydrolysed carbohydrate material.
  • each and every combination of part of the enzymatically hydrolysed carbohydrate material, part of the pretreated carbohydrate material, product of the liquefaction step and external carbon and nutrient source can be used in the culturing of the fungus.
  • the enzymatic hydrolysis comprises at least a liquefaction step wherein the carbohydrate material and/or the pretreated carbohydrate material is hydrolysed in at least a first container, and a saccharification step wherein the liquefied material is hydrolysed in the at least first container and/or in at least a second container.
  • Saccharification can be done in the same container as the liquefaction (i.e. the at least first container), it can also be done in a separate container (i.e. at least a second container). So, in the enzymatic hydrolysis liquefaction and saccharification may be combined. Alternatively, the liquefaction and saccharification may be separate steps. In an embodiment there is a solid/liquid separation between liquefaction and saccharification.
  • the enzymatic hydrolysis can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system. This also holds true when the enzymatic hydrolysis comprises a liquefaction step and a saccharification step.
  • the liquefaction step can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system and/or the saccharification step can be performed in one or more containers, but can also be performed in one or more tubes or any other continuous system.
  • Examples of containers to be used in the present invention include, but are not limited to, fed-batch stirred containers, batch stirred containers, continuous flow stirred containers with ultrafiltration, and continuous plug-flow column reactors. Stirring can be done by one or more impellers, pumps and/or static mixers.
  • the carbohydrate material and/or the pretreated carbohydrate material can be added to the one or more containers used for the enzymatic hydrolysis.
  • the enzymes used in the enzymatic hydrolysis are already present in the one or more containers before the carbohydrate material and/or the pretreated lignocellulosic material is added.
  • the enzymes used in the enzymatic hydrolysis can be added to the one or more containers.
  • the carbohydrate material and/or the pretreated carbohydrate material is already present in the one or more containers before the enzymes used in the enzymatic hydrolysis are added.
  • both the carbohydrate material and/or the pretreated carbohydrate material and the enzymes used in the enzymatic hydrolysis are added simultaneously to the one or more containers.
  • the enzymes used in the enzymatic hydrolysis may be an aqueous composition. This paragraph also holds true when the enzymatic hydrolysis comprises a liquefaction step and a saccharification step.
  • the total enzymatic hydrolysis time is 10 to 300 hours, 20 to 250 hours, preferably 30 to 200 hours, more preferably 40 to 150 hours.
  • the enzymatic hydrolysis is done at a temperature from 40°C to 90°C, from 45°C to 80°C, from 50°C to 70°C, from 55°C to 65°C.
  • the enzymatic hydrolysis is conducted until 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more of available sugar in the lignocellulosic material is released.
  • oxygen is added during the enzymatic hydrolysis. In an embodiment oxygen is added during at least a part of the enzymatic hydrolysis. Oxygen can be added continuously or discontinuously during the enzymatic hydrolysis. In an embodiment oxygen is added one or more times during the enzymatic hydrolysis. In an embodiment oxygen may be added before the enzymatic hydrolysis, during the addition of carbohydrate material to a container used of enzymatic hydrolysis, during the addition of enzyme to a container used of enzymatic hydrolysis, during a part of the enzymatic hydrolysis, during the whole enzymatic hydrolysis or any combination thereof. Oxygen is added to the one or more containers used in the enzymatic hydrolysis.
  • Oxygen can be added in several forms.
  • oxygen can be added as oxygen gas, oxygen-enriched gas, such as oxygen-enriched air, or air.
  • oxygen-enriched gas such as oxygen-enriched air
  • Examples how to add oxygen include, but are not limited to, addition of oxygen by means of sparging, blowing, electrolysis, chemical addition of oxygen, filling the one or more containers used in the enzymatic hydrolysis from the top (plunging the hydrolysate into the tank and consequently introducing oxygen into the hydrolysate) and addition of oxygen to the headspace of said one or more containers.
  • oxygen is added to the headspace of the container(s), sufficient oxygen necessary for the hydrolysis reaction may be supplied.
  • the amount of oxygen added to the container(s) can be controlled and/or varied.
  • oxygen supplied is possible by adding only oxygen during part of the hydrolysis time in said container(s).
  • Another option is adding oxygen at a low concentration, for example by using a mixture of air and recycled air (air leaving the container) or by "diluting” air with an inert gas.
  • Increasing the amount of oxygen added can be achieved by addition of oxygen during longer periods of the hydrolysis time, by adding the oxygen at a higher concentration or by adding more air.
  • Another way to control the oxygen concentration is to add an oxygen consumer and/or an oxygen generator.
  • Oxygen can be introduced, for example blown, into the liquid hydrolysis container contents of carbohydrate material. It can also be blown into the headspace of the container.
  • oxygen is added to the one or more containers used in the enzymatic hydrolysis before and/or during and/or after the addition of the carbohydrate material and/or the pretreated carbohydrate material to said one or more containers.
  • the oxygen may be introduced together with the carbohydrate material and/or the pretreated carbohydrate material that enters the hydrolysis container(s).
  • the oxygen may be introduced into the material stream that will enter the container(s) or with part of the container(s) contents that passes an external loop of the container(s).
  • the container(s) used in the enzymatic hydrolysis have a volume of at least 1 m 3 .
  • the containers have a volume of at least 1 m 3 , at least 2 m 3 , at least 3 m 3 , at least 4 m 3 , at least 5 m 3 , at least 6 m 3 , at least 7 m 3 , at least 8 m 3 , at least 9 m 3 , at least 10 m 3 , at least 15 m 3 , at least 20 m 3 , at least 25 m 3 , at least 30 m 3 , at least 35 m 3 , at least 40 m 3 , at least 45 m 3 , at least 50 m 3 , at least 60 m 3 , at least 70 m 3 , at least 75 m 3 , at least 80 m 3 , at least 90 m 3 , at least 100 m 3 , at least 200 m 3 , at least 300 m 3 , at least 400 m 3
  • the container(s) used for the liquefaction step and the container(s) used for the saccharification step may have the same volume, but also may have a different volume.
  • enzymatic hydrolysis and fermentation may be separate steps, but may also be combined.
  • examples include, but are not limited to, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF), hybrid hydrolysis and fermentation (HHF), separate hydrolysis and co fermentation (SHCF), hybrid hydrolysis and co-fermentation (HHCF), and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP).
  • SHF separate hydrolysis and fermentation
  • SSF simultaneous saccharification and fermentation
  • SSCF simultaneous saccharification and co-fermentation
  • HHF hybrid hydrolysis and fermentation
  • SHCF separate hydrolysis and co fermentation
  • HHCF hybrid hydrolysis and co-fermentation
  • DMC direct microbial conversion
  • CBP consolidated bioprocessing
  • carbohydrate material is cellulosic material, in particular lignocellulosic material.
  • Carbohydrate material suitable for use in the processes as described herein includes biomass, e.g. virgin biomass and/or non-virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper and yard waste.
  • biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane, cane straw, sugar cane bagasse, switch grass, miscanthus, energy cane, corn, corn stover, corn husks, corn cobs, corn fiber, corn kernels, canola stems, soybean stems, sweet sorghum, products and by-products from milling of grains such as corn, wheat and barley (including wet milling and dry milling) often called "bran or fibre”, distillers dried grains, as well as municipal solid waste, waste paper and yard waste.
  • the biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues.
  • Agricultural biomass includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, and hard and soft woods (not including woods with deleterious materials).
  • agricultural biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. Agricultural biomass may be any of the afore-mentioned singularly or in any combination or mixture thereof.
  • the enzyme used in the process as described herein can extremely effectively hydrolyze carbohydrate material, for example corn stover, wheat straw, cane straw, corn fiber and/or sugar cane bagasse, which can then be further converted into a product, such as ethanol, biogas, butanol, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
  • a product such as ethanol, biogas, butanol, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
  • intermediate products from a process following the hydrolysis for example lactic acid as intermediate in biogas production, can be used as building block for other materials.
  • the carbohydrate material is pretreated before and/or during the enzymatic hydrolysis.
  • Pretreatment methods are known in the art and include, but are not limited to, heat, mechanical, chemical modification, biological modification and any combination thereof. Pretreatment is typically performed in order to enhance the accessibility of the carbohydrate material to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or solubilize the hemicellulose and/or cellulose and/or lignin, in the carbohydrate material.
  • the pretreatment comprises treating the carbohydrate material with steam explosion, hot water treatment or treatment with dilute acid or dilute base. Examples of pretreatment methods include, but are not limited to, steam treatment (e.g.
  • dilute acid treatment e.g. treatment with 0.1 - 5% H 2 S0 4 and/or S0 2 and/or HN0 3 and/or HCI, in presence or absence of steam, at 120-200°C, at a pressure of 2-15 bar, at acidic pH, for 2-30 minutes
  • organosolv treatment e.g. treatment with 1 - 1 .5% H 2 S0 4 in presence of organic solvent and steam, at 160-200°C, at a pressure of 7-30 bar, at acidic pH, for 30-60 minutes
  • lime treatment e.g.
  • ARP treatment e.g. treatment with 5 - 15% NH 3 , at 150-180°C, at a pressure of 9-17 bar, at alkaline pH, for 10-90 minutes
  • AFEX treatment e.g. treatment with > 15% NH 3 , at 60-140°C, at a pressure of 8-20 bar, at alkaline pH, for 5-30 minutes).
  • the carbohydrate material may be washed.
  • the carbohydrate material may be washed before and/or after the pretreatment.
  • the washing step may be performed before and/or after solid/liquid separation of the carbohydrate material and/or the pretreated carbohydrate material. If performed after the solid/liquid separation, the solid fraction obtained after solid/liquid separation may be washed.
  • the washing step may be used to remove water soluble compounds that may act as inhibitors for the fermentation and/or hydrolysis step.
  • the washing step may be conducted in manner known to the skilled person. Next to washing, other detoxification methods do exist.
  • the pretreated carbohydrate material may also be detoxified by any (or any combination) of these methods which include, but are not limited to, solid/liquid separation, vacuum evaporation, extraction, adsorption, neutralization, overliming, addition of reducing agents, addition of detoxifying enzymes such as laccases or peroxidases, addition of microorganisms capable of detoxification of hydrolysates.
  • the pH during the enzymatic hydrolysis may be chosen by the skilled person. In an embodiment the pH during the hydrolysis may be 3.0 to 6.4.
  • a process of the invention may be carried out using high levels of dry matter (of the carbohydrate material) in the hydrolysis reaction.
  • the dry matter content of the carbohydrate material in the enzymatic hydrolysis is from 10% - 40% (w/w), 1 1 % - 35% (w/w), 12% - 30% (w/w), 13% - 29% (w/w), 14% - 28% (w/w), 15% - 27% (w/w), 16% - 26% (w/w), 17% - 25% (w/w).
  • a process for the preparation of a fermentation product from carbohydrate material comprises the steps of (a) producing a cellulolytic and/or hemicellulolytic enzyme by culturing a filamentous fungus under conditions which allow for expression of the enzyme, wherein the process comprises the step of (i) treating a sucrose- containing biomass at a temperature from 50°C to 180°C and a pH from 1 to 5 for 0.5 to 90 minutes to produce a treated biomass, (ii) culturing the filamentous fungus using the treated biomass as substrate under conditions conducive for production of the enzyme, and (iii) optionally, recovering the enzyme, (b) enzymaticall hydrolysing the carbohydrate material with the enzyme to obtain the sugar product, (c) fermenting the sugar product to produce the fermentation product, and, optionally, recovering the fermentation product.
  • the container(s) used in step (c) have a volume of at least 1 m 3 .
  • the containers have a volume of at least 1 m 3 , at least 2 m 3 , at least 3 m 3 , at least 4 m 3 , at least 5 m 3 , at least 6 m 3 , at least 7 m 3 , at least 8 m 3 , at least 9 m 3 , at least 10 m 3 , at least 15 m 3 , at least 20 m 3 , at least 25 m 3 , at least 30 m 3 , at least 35 m 3 , at least 40 m 3 , at least 45 m 3 , at least 50 m 3 , at least 60 m 3 , at least 70 m 3 , at least 75 m 3 , at least 80 m 3 , at least 90 m 3 , at least 100 m 3 , at least 200 m 3 , at least 300 m 3 , at least 400 m 3 , at least 500 m 3 , at least 600 m 3 , at least 700 m 3 , at least 800 m 3
  • the fermentation step is performed in one or more containers.
  • the fermentation can be done in the same container(s) wherein the enzymatic hydrolysis is performed.
  • the fermentation is a step in which a microorganism is used for the fermentation of a carbon source comprising sugar(s), e.g. glucose, L-arabinose and/or xylose.
  • the carbon source may include any carbohydrate oligo- or polymer comprising L-arabinose, xylose or glucose units, such as e.g. lignocellulose, xylans, cellulose, starch, arabinan and the like.
  • appropriate carbohydrases such as xylanases, glucanases, amylases and the like
  • the modified host cell may be genetically engineered to produce and excrete such carbohydrases.
  • An additional advantage of using oligo- or polymeric sources of glucose is that it enables to maintain a low(er) concentration offree glucose during the fermentation, e.g. by using rate-limiting amounts of the carbohydrases. This, in turn, will prevent repression of systems required for metabolism and transport of non-glucose sugars such as xylose.
  • the modified host cell ferments both the L-arabinose (optionally xylose) and glucose, preferably simultaneously in which case preferably a modified host cell is used which is insensitive to glucose repression to prevent diauxic growth.
  • the fermentation medium will further comprise the appropriate ingredient required for growth of the modified host cell.
  • Compositions of fermentation media for growth of microorganisms such as yeasts or filamentous fungi are well known in the art.
  • the fermentation time may be shorter than in conventional fermentation at the same conditions, wherein part of the enzymatic hydrolysis still has to take part during fermentation. In one embodiment, the fermentation time is 100 hours or less, 90 hours or less, 80 hours or less, 70 hours or less, or 60 hours or less, for a sugar composition of 50 g/l glucose and corresponding other sugars from the lignocellulosic material (e.g.
  • the fermentation time may correspondingly be reduced.
  • the fermentation time of the ethanol production step is between 10 and 50 hours for ethanol made out of C6 sugars and between 20 and 100 hours for ethanol made out of C5 sugars.
  • the fermentation time of the succinic acid production step is between 20 and 70 hours.
  • the fermentation process may be an aerobic or an anaerobic fermentation process.
  • An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed ( i.e . oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors.
  • NADH produced in glycolysis and biomass formation cannot be oxidised by oxidative phosphorylation.
  • many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD + .
  • pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1 ,3-propane-diol, ethylene, glycerol, butanol, a b-lactam antibiotic and a cephalosporin.
  • the fermentation process is anaerobic.
  • An anaerobic process is advantageous, since it is cheaper than aerobic processes: less special equipment is needed.
  • anaerobic processes are expected to give a higher product yield than aerobic processes. Under aerobic conditions, usually the biomass yield is higher than under anaerobic conditions. As a consequence, usually under aerobic conditions, the expected product yield is lower than under anaerobic conditions.
  • the fermentation process is under oxygen-limited conditions. More preferably, the fermentation process is aerobic and under oxygen-limited conditions.
  • An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
  • the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.
  • the fermentation process is preferably run at a temperature that is optimal for the modified cell.
  • the fermentation process is performed at a temperature which is less than 42°C, preferably 38°C or lower.
  • the fermentation process is preferably performed at a temperature which is lower than 35, 33, 30 or 28°C and at a temperature which is higher than 20, 22, or 25°C.
  • the alcohol fermentation step and the organic acid fermentation step are performed between 25°C and 35°C.
  • the fermentations are conducted with a fermenting microorganism, e.g. a yeast.
  • the alcohol (e.g. ethanol) fermentations of C5 sugars are conducted with a C5 fermenting microorganism.
  • the alcohol (e.g. ethanol) fermentations of C6 sugars are conducted with a C5 fermenting microorganism or a commercial C6 fermenting microorganism.
  • yeast suitable for ethanol production include, but are not limited to, BIOFERMTM AFT and XR (NABC— North American Bioproducts Corporation, GA, USA), ETFIANOL REDTM yeast (Fermentis/Lesaffre, USA), FALITM (Fleischmann's Yeast, USA), FERMIOLTM (DSM Specialties), GERT STRANDTM (Gert Strand AB, Sweden), and SUPERSTARTTM and TFIERMOSACCTM fresh yeast (Ethanol Technology, Wl, USA).
  • the alcohol producing microorganism is a microorganism that is able to ferment at least one C5 sugar. Preferably, it also is able to ferment at least one C6 sugar.
  • the application describes a process for the preparation of ethanol from carbohydrate material, comprising the steps of (a) performing a process for the preparation of a sugar product from carbohydrate material as described above, (b) fermentation of the sugar product to produce ethanol; and (c) optionally, recovery of the ethanol.
  • the fermentation can be done with a microorganism that is able to ferment at least one C5 sugar, e.g. a yeast.
  • the alcohol producing microorganisms may be a prokaryotic or eukaryotic organism.
  • the microorganism used in the process may be a genetically engineered microorganism.
  • suitable alcohol producing organisms are yeasts, for instance Saccharomyces, e.g. Saccharomyces cerevisiae, Saccharomyces pastorianus or Saccharomyces uvarum, Hansenula, Issatchenkia , e.g. Issatchenkia orientalis, Pichia, e.g. Pichia stipites or Pichia pastoris,
  • Kluyveromyces e.g. Kluyveromyces fagilis
  • Candida e.g. Candida pseudotropicalis or Candida acidothermophilum
  • Pachysolen e.g. Pachysolen tannophilus or bacteria
  • Lactobacillus e.g. Lactobacillus lactis
  • Geobacillus Zymomonas, e.g. Zymomonas mobilis
  • Clostridium e.g. Clostridium phytofermentans
  • Escherichia e.g. E. coli
  • Klebsiella e.g. Klebsiella oxytoca.
  • the microorganism that is able to ferment at least one C5 sugar is a yeast.
  • the yeast belongs to the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae.
  • the yeast, e.g. Saccharomyces cerevisiae, used in the processes according to the present invention is capable of converting hexose (C6) sugars and pentose (C5) sugars.
  • the yeast, e.g. Saccharomyces cerevisiae, used in the processes according to the present invention can anaerobically ferment at least one C6 sugar and at least one C5 sugar.
  • the yeast is capable of using L-arabinose and xylose in addition to glucose anaerobically.
  • the yeast is capable of converting L-arabinose into L-ribulose and/or xylulose 5- phosphate and/or into a desired fermentation product, for example into ethanol.
  • Organisms for example Saccharomyces cerevisiae strains, able to produce ethanol from L-arabinose may be produced by modifying a host yeast introducing the araA (L-arabinose isomerase), araB (L- ribuloglyoxalate) and araD (L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes may be introduced into a host cell in order that it is capable of using arabinose. Such an approach is given is described in W02003/095627.
  • araA , araB and araD genes from Lactobacillus plantarum may be used and are disclosed in W02008/041840.
  • the araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708.
  • araA , araB and araD genes may derived from of at least one of the genus Clavibacter, Arthrobacter and/or Gramella , in particular one of Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO 200901 1591 .
  • the yeast may also comprise one or more copies of xylose isomerase gene and/or one or more copies of xylose reductase and/or xylitol dehydrogenase.
  • the yeast may comprise one or more genetic modifications to allow the yeast to ferment xylose.
  • genetic modifications are introduction of one or more xylA- gene, XYL1 gene and XYL2 gene and/or XKSf-gene; deletion of the aldose reductase ( GRE3 ) gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate pathway in the cell.
  • GRE3 aldose reductase
  • PPP-genes TAL1, TKL1, RPE1 and RKI1 examples of genetically engineered yeast are described in EP1468093 and/or W02006/009434.
  • RN1016 is a xylose and glucose fermenting Saccharomyces cerevisiae strain from DSM, the Netherlands.
  • the fermentation process for the production of ethanol is anaerobic. Anaerobic has already been defined earlier herein. In another preferred embodiment, the fermentation process for the production of ethanol is aerobic. In another preferred embodiment, the fermentation process for the production of ethanol is under oxygen-limited conditions, more preferably aerobic and under oxygen-limited conditions. Oxygen-limited conditions have already been defined earlier herein.
  • this process is a co-fermentation process.
  • All preferred embodiments of the fermentation processes as described above are also preferred embodiments of this co-fermentation process: identity of the fermentation product, identity of source of L-arabinose and source of xylose, conditions of fermentation (aerobic or anaerobic conditions, oxygen-limited conditions, temperature at which the process is being carried out, productivity of ethanol, yield of ethanol).
  • Fermentation products that may be produced by the processes of the invention can be any substance derived from fermentation. They include, but are not limited to, alcohol (such as arabinitol, butanol, ethanol, glycerol, methanol, 1 ,3-propanediol, sorbitol, and xylitol); organic acid (such as acetic acid, acetonic acid, adipic acid, ascorbic acid, acrylic acid, citric acid, 2,5-diketo-D- gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3- hydroxypropionic acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); ketones (such as acetone); amino acids (such as aspartic acid, glutamic acid
  • the fermentation product can also be a protein, a vitamin, a pharmaceutical, an animal feed supplement, a specialty chemical, a chemical feedstock, a plastic, a solvent, ethylene, an enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductase, a transferase or a xylanase.
  • an alcohol is prepared in the fermentation processes as described herein.
  • ethanol is prepared in the fermentation processes as described herein.
  • the processes as described herein may comprise recovery of all kinds of products made during the processes including fermentation products such as ethanol.
  • a fermentation product may be separated from the fermentation broth in manner know to the skilled person. Examples of techniques for recovery include, but are not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For each fermentation product the skilled person will thus be able to select a proper separation technique. For instance, ethanol may be separated from a yeast fermentation broth by distillation, for instance steam distillation/vacuum distillation in conventional way.
  • a Rasamsonia strain was cultivated in a preculture shake flask (300 ml) for 3 days at 45°C and 200 rpm.
  • the medium used for cultivation comprised yeast extract powder (20 g/l), dextrose (22 g/l) and MES hydrate (20 g/l). At the end of the cultivation time, all dextrose was consumed. Subsequently, 1 ml of the content of the preculture shake flask was transferred to each of the two shake flasks in the below experiment.
  • the Rasamsonia strain was cultured in two shake flasks (50 ml) for 4 days at 45°C and
  • the hydrolyzed sucrose solution was prepared by treating the 3.8% (w/w) sucrose solution for 20 minutes at a temperature of 121 °C and a pH of 3.
  • the pH of the 3.8% (w/w) sucrose solution was brought to pH 3 by using phosphoric acid.
  • the cumulative oxygen transfer during culturing was measured by the equipment "Ramos from Kiihner”. The cumulative oxygen transfer after 65 hours of culturing is shown in Table 1 .
  • the cumulative oxygen transferred by the Rasamsonia strain grown in the shake flask with hydrolyzed sucrose was about 14 times higher than in the shake flask with untreated sucrose.
  • An increased cumulative oxygen transfer rate reflects increased biomass formation and increased biomass formation leads to increased production of cellulolytic and/or hemicellulolytic enzymes.
  • a Rasamsonia strain (strain TEC-210 as described in WO 201 1/000949) was cultivated in shake-flasks (300 ml) for 3 days at 45°C and 200 rpm. During this cultivation, both biomass is formed and proteins are produced.
  • the cultivation medium was based on carbon source (20 g/kg), yeast extract (20 g/kg), MES hydrate (20 g/kg), and cellulose (1 .5 g/kg).
  • the carbon source sucrose
  • the pH at the start of cultivation was about 5.2.
  • To one shake-flask 100 ml 6.0% (w/w) sucrose solution was added and to one shake-flask 100 ml 6.0% (w/w) hydrolyzed sucrose solution was added.
  • the hydrolyzed sucrose solution was prepared by pretreatment of the 6.0% (w/w) sucrose solution.
  • the pH of the sucrose solution was decreased to 3 using hydrochloric acid and afterwards the sucrose solution was incubated for 20 minutes at 121 °C.
  • Biuret test In the Biuret reaction, a copper (II) ion is reduced to copper (I), which forms a complex with the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color, and hence the absorption at 546 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.
  • BSA Bovine Serum Albumine
  • the standardisation was performed using BSA (Bovine Serum Albumine) and the protein content was expressed in g protein as BSA equivalent/L or mg protein as BSA equivalent/ml.
  • the protein content was calculated using standard calculation protocols known in the art, by plotting the OD546 versus the concentration of samples with known concentration, followed by the calculation of the concentration of the unknown samples using the equation generated from the calibration line.
  • Sucrose was determined by HPLC-RI using a Rezex RNM-
  • Biomass formation Biomass formation was determined by dry weight analysis using the filtration method. A pre-weighed glass microfiber filter (particle retention: 1 .6 pm) was placed on a Biichnerfunnel. Vacuum was applied, and a pre-weighted amount of broth was slowly filtered. Next a washing step was performed wherein plenty of demi-water was used (about 3x the applied broth weight).
  • Biomass formation and protein production of the shake-flask wherein hydrolyzed sucrose was used as carbon source were 40% higher (biomass) and 74% higher (protein), respectively, compared to the shake-flask with untreated sucrose as carbon source.
  • the results were supported by the fact that almost all sucrose was left over in the shake flask with untreated sucrose as carbon source and all sucrose was converted in the shake flask with hydrolyzed sucrose as carbon source.
  • a Rasamsonia strain (strain TEC-210 as described in WO 201 1/000949) was cultivated in shake-flasks (300 ml) for 3 days at 45°C and 200 rpm. During this cultivation, biomass is formed and proteins are produced.
  • the cultivation medium was based on carbon source (20 g/kg), yeast extract (20 g/kg), MES hydrate (20 g/kg), and cellulose (1 .5 g/kg).
  • the carbon source (sucrose) was added separately to the shake flask.
  • the pH at the start of cultivation was about 5.2.
  • To two reference shake-flasks 100 ml 6.0% (w/w) sucrose solution was added and to the other shake- flasks 100 ml 6.0% (w/w) hydrolyzed sucrose solution was added.
  • the hydrolyzed sucrose solution was prepared by pretreatment of the 6.0% (w/w) sucrose solution. A combination of pretreatment conditions was applied. For specific pH, temperature and incubation times see Tables 3 and 4. The pH of the sucrose solution was decreased to 5.0, 3.0 or 1 .7 using hydrochloric acid and a calibrated pH electrode. Next, the sucrose solution was incubated for 5 minutes or 60 minutes at 50°C, 105°C or 180°C by putting the solution in an oven incubator.
  • biomass formation was measured in the shake flasks (see Table 3). Biomass formation was determined by measuring the dry weight of the broth as described in Example 2. At the end of cultivation, also protein production was determined (see Table 4). This was done using the Biuret test as described in Example 2.
  • Biomass formation and protein production of the shake-flasks wherein hydrolyzed sucrose was used as carbon source were at least 1 1 % higher (for biomass) and at least 8% higher (for protein), respectively, compared to the average of the shake-flasks with untreated sucrose as carbon source.
  • Tables 3 and 4 show that higher biomass formation and higher protein production were found for various different pretreatment condtions.
  • Table 1 Cumulative oxygen transferred after 65 hours of culturing.

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Abstract

L'invention concerne un procédé de production d'un polypeptide par culture d'un micro-organisme fongique filamenteux sur une biomasse contenant du saccharose, le saccharose étant d'abord hydrolysé dans des conditions acides pour améliorer la croissance globale du micro-organisme, ce qui améliore également le rendement enzymatique.
PCT/EP2019/062608 2018-05-17 2019-05-16 Procédé de production d'un polypeptide WO2019219804A1 (fr)

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