FERMENTABLE SUGARS WITH INTEGRATED ENZYME

PRODUCTION FIELD OF THE INVENTION

The present invention relates to a process for combined enzyme production and biomass hydrolysis. In particular, it relates to a process of producing cellulolytic enzymes using a genetically engineered fungus on a medium comprising sugar derived from biomass hydrolysis and carrying out biomass hydrolysis in a single reaction vessel. The present invention also provides suitable genetically modified microorganisms that enable the successful application of the disclosed process.

BACKGROUND OF THE INVENTION

Lignocellulosic biomass is a term used to describe the recalcitrant structural plant material that is inedible to humans. Commonly cited examples of lignocellulosic biomass include wood, cereal straw, corn stover and sugarcane bagasse. Being abundant, low-cost, geographically relatively evenly distributed, chemically well-defined and renewable, it is seen as an attractive raw material base for the production of fuels and chemicals in the future. In particular, it is seen as one of the only viable renewable alternatives to petroleum in what is commonly referred to as bioeconomy. ,

Dry lignocellulosic biomass comprises mainly plant cell walls, which are made up of three chemically distinct fractions: cellulose, hemicellulose and lignin. Cellulose and hemicellulose are polysaccharides, i.e. polymers of repeating sugar units. Cellulose, the primary component of biomass representing up to 50% of dry-weight, is a homopolymer of repeating units of D-glucose linked by 3-1,4-glycosidic bonds. These cellulose chains further assemble into larger cellulose fibers primarily through hydrogen bonding between individual chains. Hemicellulose typically represents between a third and a fifth of dry-weight, is more complex and its precise composition varies depending on the botanical origin of the material/The bulk of hemiceilulose in hardwoods and grasses consists of xylan, a polymer of the pentose sugar xylose, while softwoods additionally contain large amounts of mannan, a polymer of the hexose sugar mannose.

Hemiceilulose also typically contains lesser amounts of other sugars such as arabinose, and organic acids such as acetic and ferulic acid. The remaining fraction of lignocellulosic biomass - lignin - is a complex, randomly assembled polyphenol providing rigidity to plant structure.

Two industrial routes from lignocellulosic biomass to renewable fuels and chemicals are commonly discussed: the thermochemical and biochemical routes. Thermochemical processing methods include pyrolysis and gasification, which use high temperatures and/or pressures to decompose biomass into pyrolysis oil or syngas that can further be refined into desired products. The biochemical route, on the other hand, seeks to maintain the basic chemical building blocks, particularly the sugars, of lignocellulosic biomass intact so they may be fermented into final products. The favored method for the extraction of sugars from lignocellulosic biomass is enzymatic hydrolysis. In this process, enzymes - protein biocatalysts produced by microbes - are used to break down the polysaccharide components cellulose and hemiceilulose into their soluble constituent sugars.

Several enzymes are known to act in the process of hydrolyzing biomass polysaccharides, and these enzymes can be classified in different ways. One useful way to classify enzymes is by their specificity, i.e. by the kinds of substrates on which they act. Enzymes acting on cellulose include ceilobiohydrolases (E.C. 3,2.1.91), endoglucanases (E.G. 3.2.1.4) and β- glucosidases (3.2.1.21). Together these enzymes are known by the general term of "cellulase". More recently, non-hydrolytic enzymes participating in the breakdown of cellulose have been discovered, such as swollenins and lytic polysaccharide monooxygenases (1). A wide variety of enzymes also participates in the hydrolysis of hemiceilulose. Some of the most notable of these enzymes include xylanase (E.C. 3.2.1.8), β- xylosidase (E.C. 3.2.1.27), mannanase (E.C. 3.2.1.28), arabinofuranosidase (E.C. 3.2.1.55) and acety!-xylan esterase (3.1.1.72). Enzymes degrading hemicellulose are known by the general term

"hemiceilulase" (2).

Compared to other established industrial enzyme applications, such as the liquefaction of starch using amylase, the hydrolysis of lignocellulosic polysaccharides requires far greater doses of enzymes. Typically, the enzyme loadings in this type of hydrolysis are in the range of 0.1 to 2 wt% of the substrate. This high dose requirement is largely a consequence of the recalcitrant nature of the lignocellulosic substrate. Both starch and cellulose are polymers of glucose, but whereas starch is produced by plants as a rapidly available storage-form of energy in seeds and tubers, cellulose is synthesized to provide structural support and has the properties of a crystal. For industrial applications involving cellulose hydrolysis, this in turn means that the enzymes impart a large operational cost (3).

The favored organism for the production of (hemi) cellulase enzymes has traditionally been the mesophilic ascomycete fungus Trichoderma reesei (Hypocrea jecorina). This fungus was isolated from the Solomon Islands during the Second World War and in the following decades became the reference organism for cellulase enzyme production. Albeit having a genome encoding relatively few glycoside hydrolases (4), the enzymes produced by 7. reesei are seen as particularly efficient in the hydrolysis of crystalline cellulose (5). Consequently, this fungus has been under intensive investigation and a large number of strains have been developed for different applications. Cellulase production was originally improved by random mutagenesis, resulting in well-known strains such as QM941 and Rut-C30, which secrete more than ten times greater amounts of enzymes than the wild type strain QM6a (6).

However, even a T. reesei strain improved by mutagenesis is seen as unable to support economically viable enzymatic degradation of biomass for industrial applications. Although strains such as Rut-C30 are considered derepressed, meaning that cellulase production is not inhibited by glucose, inducing compounds such as lactose, sophorose or pure cellulose are still required for enzyme production. High costs are associated with culture media containing these ^* inducing compounds and the specialized equipment required for fungal fermentation.

Although efficient at degrading crystalline cellulose, the enzymes produced by T. reesei are obviously lacking at least with respect to β- glucosidase activity and their performance can further be improved by including other auxiliary enzymes. Due to the great economic potential of cellulose hydrolysis, there have been continuous efforts to improve the performance of production strains, enzymes and processes to enable a cost-competitive industrial route to fermentable sugars. Three primary approaches to making enzymatic cellulose hydrolysis viable can be defined: improving enzyme performance, increasing enzyme production by the production strains and reducing the cost of the enzyme.

So far, perhaps most success has been achieved in improving the performance of cellulolytic enzyme mixtures, particularly by leading enzyme companies. A notable drawback of the enzymes naturally produced by T. reesei is the low level of β-glucosidase secreted by this fungus (7). This enzyme acts in the final step of cellulose hydrolysis, cleaving the disaccharide cellobiose into two glucose units. The step is very important as final reaction products inhibit the enzymes acting in previous steps and thus slow down the entire process.

For the hydrolysis of biomass, 7 ^" . reesei enzymes were originally supplemented with β-glucosidase produced by another organism, typically Aspergillus niger. However, T. reesei itself carr be genetically engineered to produce more of this key enzyme, something that has been

demonstrated in several studies (8-10). Enzymes produced by T. reesei have also been improved by applying enzyme engineering to

cellobiohydrolases, endoglucanases and xylartases, and by expressing better heterologous enzymes from other organisms. Thus, the application WO2011098551 describes improved cel!obiohydrolases (11 ) and

WO2010135836A1 improved β-glucosidases (12). Novel types of enzymes, particularly polysaccharide monooxygenases, have also been added to the enzyme mixtures to improve hydrolysis performance. All these improvements have meant reduced enzyme loading requirements for commercial enzymes (3).

Although T. reesei strains improved by mutagenesis, such as

QM9414 or RutC30, can under the right conditions be used to produce great amounts of cellulase, measured in tens of grams per liter, there remains room for improvement. Enzyme productivity and yield can be further improved by genetic engineering. An important means to this end is the modification of transcription factors controlling enzyme secretion in T. reesei. Several such transcription factors have been described in T.

reesei, traditionally Cre1 , ACE1 , ACE2, Hap2/3/5 and Xyr1 (13) but more recently also others (14). Overexpression or deletion of given transcription factors has been shown to increase enzyme production by T. reesei Rut- C30 (15). Thus, application WO2010060188 describes the use of T. reesei strains overexpressing Xyr1 with growth media rich in hemicellulose- derived sugars (16) and WO20 1151513A1 describes a novel

transcription factor for improved enzyme production (17).

A final approach to enabling the cost-efficient enzymatic hydrolysis of lignocellulose is to reduce the production cost of the enzyme. This could be achieved, for example, by growing the enzyme-producing fungus on low-cost raw materials. According to some estimates, over 50% of the enzyme cost is derived from the carbon source that is fed to the fungus, even if that carbon source were glucose (18). However, to reach high enzyme titers with conventional T. reesei strains, expensive inducing carbon sources such as lactose, sophorose or macrocrystalline cellulose are required.

The production of enzymes at their final end-site of use ("on-site" enzyme production) has been presented as an option to reduce enzyme costs (19). Commercial enzymes are presumably produced on relatively pure culture media components, filtered, processed and delivered to customers in trucks, and the associated costs could be avoided if the enzymes were produced on-site instead. Ideally, enzyme production would use material streams from the process itself. The patent application WO 2012089844A1 describes a process where celiulases are produced on- site using pre-treated biomass as carbon source and then used in hydrolysis along with the fungal mycelia (20).

Alternatively, fungi could be used to produce the necessary enzymes on the substrate biomass in the same vessel where hydrolysis takes place (21-23). However, it is highly doubtful if such a set-up would be applicable at the high biomass concentrations (20 wt% or more) used in industrial scale enzymatic hydrolysis. The use of biomass for enzyme production faces notable hurdles: Biomass substrates are' highly viscous and would need to be used at prohibitive concentrations for desired enzyme yields. In comparison with soluble carbohydrates, biomass is also consumed considerably more slowly by fungi, extending enzyme production times. The lignin present at high ratios in biomass substrates causes additional problems: It is not significantly consumed by soft-rot fungi and would therefore accumulate and cause problems in the subsequent hydrolysis step. Lignin also has the disadvantage of binding and inactivating celiulases (24).

Only a part of the carbon found in in-natura biomasses is readily available to fungi and fungi are highly susceptible to the inhibitors present in pre-treated biomasses. Sugar-containing liquid streams of the cellulosic ethanol process would appear to provide another possible locally available carbon source. However, neither of these streams alone induces cellulase production in conventional fungal strains. More critically, these streams also contain high concentrations of fermentation inhibitors. Very high concentrations of inhibitors and low concentrations of sugars are found in liquid hemicellulosic fractions, which are produced in pre-treatment processes. Enzymatic biomass hydrolysates, although containing slightly less inhibitors and more sugars, are still too toxic to be used at the very high concentrations required in conventional enzyme production. So, although both of these sugar streams are mentioned as potential carbon sources in other documents (16, 25), no explanation is given how the effect of fermentation inhibitors contained in these streams would be overcome.

To the best knowledge of the inventors, only delivered commercial enzymes have so far found industrial-scale application. There therefore remains a need in the art for the cost-efficient production of cellulases at their end-site of use. In particular, there is a need in the art for an industrially feasible, economical, simple approach to the hydrolysis of lignocellulosic biomass that would limit the operational cost imposed by enzyme production. ·

SUMMARY OF THE INVENTIO

The present invention relates to a simplified process for enzyme production and hydrolysis. Provided is a process where enzyme

production and biomass hydrolysis are performed sequentially in a single vessel. The invention provides a closed-loop model, where the carbon source for enzyme production is a stream of process itself. The disclosed invention is based on the observation that biomass hydrolysate may be used as the primary carbon source for production of hydrolytic enzymes on-site, when a suitable genetically modified fungus is employed and the hydrolysate is sufficiently diluted to avoid the toxic effect of inhibitors. Provided in this invention are suitable genetically modified fungal strains and methods for producing suitable genetically modified fungal strains that enable the application of the described process. The low concentration of carbon source used and the low concentration? of enzymes consequently produced favor performing all process steps in a single reaction vessel.

The disclosed circular process contains two or more distinct steps that are performed sequentially within the same vessel. Defining steps of the disclosed process are a) an enzyme production step using a genetically engineered fungus on a dilute medium comprising primarily biomass hydrolysate resulting from step b) of the process, and b) a hydrolysis step where biomass is added into the vessel so that the polysaccharide components of the biomass may be hydrolyzed by the enzymes produced by the fungus in step a) of the process.

The fungus used in the enzyme production step (step a) of the process is genetically modified so that it overexpresses 1) at least one transcription factor and 2) at least one cellulase. The overexpression of a transcription factor has the effect of increasing overall enzyme production under the cultivation conditions used in step a) of the process (enzyme production) when compared to a parental strain lacking the modification. The overexpression of a cellulase has the effect of improving the performance of the enzymes produced by the fungus in step b) of the process (hydrolysis) when compared to a parental strain lacking the modification. These two genetic modifications are considered the minimum modifications necessary for the successful application of the disclosed process.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - Schematic illustration of the preferred embodiment of the disclosed process

Figure 2 - Schematic illustrations of the DNA vectors pVTTBR43 and pVTTBR54 used to create the T. reesei strains VTT-BR-C0019 and - C0020, respectively.

Figure 3 - Extracellular enzyme (A) and β-glucosidase (B) production by parental strain VTT-BR-C0001 (circles) and derived strains VTT-BR- G0019 (squares) and -C0020 (triangles) in shake flasks using an inducing (filled symbols) and repressing (empty symbols) carbon source.

Figure 4 - Enzyme profile of strains VTT-BR-C0001 , -C0019 and - C0020 cultured on either an inducing (Avicel+iactose) or repressing (glucose) carbon source.

Figure 5 - Hydrolysis of pre-treated sugarcane bagasse using the enzymes produced by T. reesei strains VTT-BR-C0001 (circles), -C0019 (squares) and -C0020 (triangles) on inducing (filled symbols) and repressing (open symbols) media. A. Reducing sugars released from the bagasse by each enzyme as a function of time and B. Glucose, xylose and cellobiose measured from the final hydrolysates produced by each enzyme.

Figure 6 - Results from a laboratory bioreactor test (Example 5) producing enzymes using T. reesei strain VTT-BR-C0020 with glucose and fructose derived from sugarcane molasses as carbon source and performing biomass hydrolysis in the same bioreactor.

Figure 7 - Results from a laboratory bioreactor test (Example 6) using T. reesei strain VTT-BR-C0020 for enzyme production, biomass hydrolysate as carbon source and S. cerevisae for ethanol fermentation according to the process scheme depicted in Figure 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a combined process for enzyme production using a genetically modified fungus and biomass hydrolysis performed in a single reaction vessel. The disclosed process contrasts with conventional enzyme production, which is performed in dedicated enzyme production vessels from which the enzymes are transferred for use to separate hydrolysis vessels. This single-vessel approach allows the use of large enzyme volumes relative to the b mass substrate without high associated costs. Enzyme concentrations may therefore be low, and low concentrations of carbon sources may be ¾ised for their production. Carbon sources that would be toxic at nigh concentrations consequently become viable options under the proposed process conditions.

The process also contrasts with previously suggested single-vessel approaches to enzyme production and biomass hydrolysis in not utilizing biomass as the substrate for enzyme production. Instead, the enzymes are produced using biomass hydrolysate as the primary carbon source. The sugars contained in biomass hydrolysates are epressors and do not induce cellulase production in most fungi. A genetically engineered fungus is therefore required for the successful application of the disclosed invention. A suitable genetically engineered fungus is provided in this invention.

The process described in this invention comprises at least the two fundamental steps of: a) inoculating a dilute liquid culture medium comprising biomass hydrolysate as the primary carbon source in a vessel with a genetically modified fungus and at a later point b) adding biomass into the same vessel, with the purpose of hydrolyzing its polysaccharide components into soluble sugars using the enzymes produced by the fungus in step a).

With "vessel" as used herein is meant a physically confined space in which the process takes place. The vessel may be composed of glass, steel or other any other material. It may be a closed container, but may contain openings for addition and removal of -materials. Materials that can be added to the vessel include liquid growth media, carbon sources, microbial cells, acids, bases, antifoams, compressed air, oxygen, nitrogen and other gasses. Materials that may be removed from the vessel include process samples, lignin, insoluble carbohydrate, soluble sugars, gasses and fermentation products. The vessel may provide a means of agitating the internal contents, such as rotors, upward gas flows or mechanisms for the agitation or rotation of the vessel itself. The vessel may also provide a means of sterilizing the contents of the vessel, such as the possibility of increasing temperature above 80°C and/or pressure above atmospheric pressure.

Step a) of the process is known as "enzyme production" and is performed in the vessel using a dilute culture medium that is rapidly consumed by the genetically engineered fungus. The carbon source used to grow the fungus and produce enzymes comprises primarily enzymatic biomass hydrolysate resulting from step b) of the process. Other low-cost carbon sources can optionally be included in enzyme production. The carbon source(s) may be present in the reacto? since the moment of inoculating the fungus (batch fermentation), or it may be fed into the reactor during cultivation (fed-batch fermentation). In addition to carbon sources, the medium may contain suitable nitrogen sources, phosphorus sources, and essential salts. ^*

The concentration of the carbon source or sources relative to the final volume of the culture medium used for enzyme production may be from about 0.1 to about 10% (w/v), or any concentration there between. More preferably, the concentration of the carbon source or carbon sources may be from about 0.5 to about 5% (w/v), and even more preferably from about 1.0 to about 3 % (w/v) or any concentration there between. Of the total carbon source used, biomass hydrolysate represents from about 50 wt% to about 100 wt%. More preferably, biomass hydrolysate represents from about 60 wt%, from about 70 wt%, from about 80 wt% or from about 90 wt% to about 100 wt% of the total carbon source used.

The enzyme production step is initiated by adding a cell-inoculum of the genetically engineered fungus into the vessel. The cell-inoculum of the genetically engineered fungus is produced in a vessel smaller than the vessel used for enzyme production and hydrolysis. The smaller vessel may form part of what is known as a seed-train, or a series of vessels of increasing volume. The culture medium used to produce the cell-inoculum may be the same as used for enzyme production in the vessel or it may be different. The cell-inoculum added to the enzyme production vessel may contain an amount of cells with a dry-weight fr¾m about 0.1 g/L to about 100 g/L or any value there between and the diV-weight of cells in the enzyme production vessel after inoculation may be from about 0.01 g/L to about 10 g L or any value there between. Methods for measuring the dry- weight of cells from a liquid sample are well known to those skilled in the art. The pH during enzyme production can be from about 2.0 to about 7.0, more preferably from about 3.0 to about 6.0 and more preferably from about 4.0 to about 5.0. The temperature can be from about 5°C to about 45°C, more preferably, from about 15°C to about 35°C and more preferably from about 20°C to about 30°C.

The fungus may rapidly consume the dilute culture medium in step a) and produce low concentrations of extracellular enzymes. The enzyme production step may take from about 1 hour to about 150 hours, more preferably from about 5 to about 100 hours and more preferably from about 0 to about 50 hours and enzyme concentrations at the end of the first step can be from about 0.5 g/L to about 20 g/L or any value there between.

Step b) of the disclosed process is known as "hydrolysis", and is considered to begin with the addition of biomass substrate into the reactor. Preferred biomass substrates include sugarcane bagasse, sugarcane straw, corn stover, wheat straw, barley straw, rye straw, rice straw, sorghum straw, wood chips, sawdust, switchgrass (Panicum virgatum), Giant Cane (Arundo donax), silvergrass (Miscanthus spp.) and empty palm fruit bunches. The biomass substrate is optionally pre-treated. Pre- treatments that may be employed include physical, thermal, chemical or other type of processes that modify the structure of biomass in a way that it becomes more susceptible to enzymatic degradation. Biomass addition to the reactor can be performed in one step, of the addition may be stepwise or it may be continuous.

The biomass fed into the reactor can have a dry-weight content of about 20 wt% to about 80 wt%. More preferably, the pre-treated biomass has a dry-weight of about 30 wt% to about 70 wt% and more preferably from about 40 wt% to about 60 wt%; and final solids content in the reactor after the addition of all the biomass substrate may be from about 10 wt% to about 40 wt%. More preferably, the solids content is from about 15 wt%o about 35 wt%, and even more preferably frcm about 20 wt% to about 30 wt%. The contents of the vessel may be agitated by the same means as used during "enzyme production" or by any other means.

During step b) of the process (hydrolysis) the fungus from step a) is prevented from consuming over 50% of the sugars that are released from the biomass. The released sugars may either accumulate in solution or they may be simultaneously consumed by another organism or other organisms added into the vessel before or during step b). Hydrolysis can be carried out at a temperature from about 20°C to about 70°C, more preferably at a temperature of about 30°C to about 60°C and more preferably at a temperature of about 40°C to about 50°C. The hydrolysis step may take from about 6 hours to about 129 hours or any amount of time there between. More preferably, hydrolysis is carried out for about 12 hours to about 90 hours and more preferably for around 24 hours to around 72 hours.

If no other organism is added to the reactor before or during the second step, the total concentration of soluble single sugars, such as but not limited to glucose, xylose, arabinose, galactose and mannose may accumulate in solution to concentrations from about 40 to about 250 g/L. More preferably the sugars accumulate to concentrations from about 60 to about 200 g/L and even more preferably to concentrations from about 80 g/L to about 150 g/L. The solution containing the sugars may be separated from the solid residues of the process, including lignin and cell mass, by methods such as centrifugation and filtration.

Provided in the invention is a suitable furtgal strain to be used in step a) of the process that is genetically modified m a way that it overexpresses ) at least one transcription factor and 2),at least one cellulase.

The fungus used in step a) of the process may belong to the genera Trichoderma, Hypocrea, Aspergillus, Penicillium, Rhasamsonia,

Talaromyces, Myceliophtora, Neurospora or Humicola.

The overexpression of a transcription factor is such that it increases enzyme production by the fungus in step a) of the process when compared to a parental strain lacking the modification. The overexpression of a cellulase is such that the performance of the enzymes produced in step a) of the process is improved in step b) of the process when compared to the enzymes produced by a parental strain lacking the modification. These two modifications are considered necessary for the successful application of the process. ··

The transcription factor may be a native or modified Trichoderma reesei Cre1 , ACE1 , ACE2, ACE3 or Xyr1 transcription factor or the closest homologue thereof in Trichoderma, Hypocrea, Aspergillus, Penicillium, Rhasamsonia, Talaromyces, Myceliophtora, N urospora or -Humicola. Overexpression of the transcription factor may be constitutive or inducible.

The overexpressed cellulase may be a cellobiohydrolase, an endoglucanase or a β-glucosidase. The cellulase in question may be native or heterologous with respect to the host fungus. Overexpression of a cellulase may be constitutive or inducible.

The fungal strain used in step a) may contain additional genetic modifications in addition to those previously mentioned. Such

modifications may involve the deletion, modification or overexpression of an invertase, a transcription factor, a cellulase, a hemicellulase, a swollenin, a polysaccharide monooxygenase or a cellobiose

dehydrogenases.

Genetic engineering

With "genetically modified" is meant that |he genetic material of the organism in question has been altered using genetic engineering

techniques. The genetic material is composed of deoxyribonucleic acid (DNA) and may be nuclear, chromosomal, episomal or mitochondrial in nature. The "genetic modification" may be a mutation, insertion or deletion of genetic material.

A "genetically modified fungus" is a fungslstrain whose genetic material has been modified using suitable techniques. One skilled in the art knows that a linearized DNA fragment may be transformed into a fungal cell, whereby it may integrate into the genome of the organism and in so doing make the fungai cell "genetically modified". A linearized DNA fragment may be produced, for example, by digesting a plasmid with a suitable restriction enzyme.

With "transformation" is meant the process by which genetic material passes the cell membrane and becomes incorporated in the organism. Transformation of fungi may be carried out using methods such as protoplast transformation (26) or by biolistic botnbardment (27). To select for successful transformants, so-called marker genes may be included in the linearized DNA used to transform the fungus. One skilled in the art knows that suitable marker genes for transformation of filamentous fungi include those encoding acetamidase (amdS), hygromycin

phosphotransferase (hph), orotodine 5'-phosphate carboxylase (pyrG) and phosphinothricin acetyltransferase (pat). Integration of a DNA fragment into the genome of a transformant can be verified using techniques familiar to those skilled in the art, such as PCR, Southern blotting and DNA sequencing.

As used herein, with "parental strain" is meant a strain of a fungus where a given genetic modification has not been made, but that is otherwise identical to the genetically modified fungus.

A "transcription factor" describes a protein that binds to specific DNA sequences and controls the expression of adjacent genes. A transcription factor may activate or repress the expression sf the adjacent genes and it may do so alone or in conjunction with other t anscription factors or other proteins.

With "overexpression" as used herein is meant any modification that increases the expression of the gene in question relative to the parental strain. Those skilled in the art know that overekpression can be brought about by adding additional copies of a gene or changing the promoter of a gene, or by other suitable means. Overexpression may be verified by measuring the concentration of the RNA or protein product of a gene. Methods for quantifying an RNA-species are well known to those skilled in the art and include Northern blotting, quantitative PCR and RNAseq. Methods for quantifying the concentration of a specific protein are also provided for in the art, the most common being Western blotting. In the case of enzymes, overexpression can also be verified by measuring the increase in the corresponding activity brought about by the

overexpression. The gene is considered overexpressed if the

concentration of the RNA or protein product of the gene is higher in the genetically modified strain than in the parental strain under equal conditions of cultivation. As used herein, the expression of a heterologous or modified sequence is by definition considered overexpression, as the gene product is lacking in the parental strain.

As used herein, "increasing enzyme production" means an increase in the concentration of extracellular enzymes produced by the fungus under identical cultivation conditions in an equal amount of time. Such an increase can be quantified by measuring the total amount of proteins in the cultivation supernatant. Methods for such quantification are well known to those skilled in the art and include those described by Lowry (28) and Bradford (29). Fungal cultivation supernatants may contain compounds other than proteins that may interfere with protein quantification.

Therefore, it may be advantageous to precipitate proteins from a supernatant before quantification. In the art are described methods for precipitating protein from solution, such as precipitation using acetone or trichloroacetic acid (TCA).

The overexpression of a transcription factor may involve a

Trichoderma Cre1 , Ace1 , Ace2, Ace3, Xyr1 or Hap2/3/5 transcription factor or the closest homologue thereof in Triefioderma, Hypocrea, Aspergillus, PenicHlium, Rhasamsonia, Talaromyces, Myceliophtora, Neurospora or Humicola.

As used herein, "constitutive" is used to refer to a form of gene expression that is constant under various types of cultivation conditions. With "inducible" is meant a form of gene expression that requires an inducing factor and is not constant. The inducing factor may be a molecule or a change in physical cultivation parameters such as temperature.

With "closest homologues" as used herein is meant a gene in another fungus whose nucleotide sequence has the highest percentage of identical nucleotides with the Trichoderma transcription factor gene; or a gene whose protein product has the highest number of identical amino acids with the protein product of the Trichoderma transcription factor gene of all the genes in an organism. Methods for identifying such closest homologue genes are well known to those skilled in the art, and include the Basic Local Alignment Search Tool (BLAST) (30).

In a preferred embodiment of the invention, the modification relating to a transcription factor is an overexpression of a T. reesei Xyr1

transcription factor with a mutation of a valine -residue at position 821 to a phenylalanine (V821 F) (SEQ ID N01). With a "residue" here is meant an amino acid that has been linked to other amino acids by peptide bonds forming a polypeptide. With the "position" of a residue is meant the sequential number of the amino acid residue when counting from the end of the polypeptide chain containing a free amino-group (the N-terminus). In the preferred embodiment, the transcription factor is under the control of a pyruvate decarboxylase (pdc) promoter.

With "cellulase" is meant a protein that has hydrolytic activity on cellulose or cello-oligosaccharides. The enzyme may be a

cellobiohydrolase (E.C. 3.2.1.91), an endoglucanase (E C. 3.2.1.4) or a β- glucosidase (E.C. 3.2.1.21). Methods for determining if a protein is a cellobiohydrolase, an endoglucanase or β-glucosidase are well known to those skilled in the art. As used herein, the protein is considered a

"cellulase" if it displays significant activity against one of the- substrates selected from the group: Filter paper, Avicel, carboxymethy!celiuiose, hydroxyethylcellulose, celSohexaose, cellopentose, cellotetraose, cellotriose, cellobiose, 4-n¾rophenyl- -D-glucopyran0side, 4-nitrophenyl- - D-cellobioside and 4-methylumbelliferyl-P-D-lastopyranoside. The cellulase may be native to the host fungus or it may be heterologous. With "heterologous" is meant that the enzyme is originally from another species.

With "improved performance of enzymes" is meant that under identical reaction conditions the enzyme liberates more soluble sugars from a pre-treated biomass substrate in the same amount of time. With "reaction conditions" are meant temperature, ρΉ, substrate concentration, agitation and enzyme dosage. The released soluble sugars can be quantified by methods known to those skilled in the art, which include the DNS method (30), using enzymatic assay kits such as those employing glucose oxidase, or using high-performance liquid chromatography

(HPLC).

In a preferred embodiment, the overexpressed cellulase is a Cel3A beta-glucosidase from T. emersonii (SEQ ID ΝΘ2) under the control of the inducible Xyn11 B xylanase promoter of T. reesei.

Enzyme production

With "enzyme production" is hereby meant a submerged

fermentation process in which a microorganism is cultivated in a liquid medium with the primary aim of producing enzymes. The microorganism can be a fungus belonging to the subdivision Pezizomycotina. Preferred, non-limiting examples include the genera Trichoderma, Hypocrea,

Aspergillus, Talaromyces, Rhasamsonia, Myceliopthora, Penicillium, Humicola and Neurospora. In the preferred embodiment, the

microorganism is a genetically modified fungus of the genera Trichoderma or Hypocrea.

The dilute liquid medium used in the enzyme production step may contain suitable compounds allowing the growth of the fungus and the production of enzymes by the fungus. Suitable compounds are known to those skilled in the art, and include macronutrisnts such as carbon sources, nitrogen sources and phosphorus soarces, and essential salts. Suitable carbon sources include single sugars such as glucose, fructose, mannose, xylose, galactose and arabinose, poiyols such as xylitol, mannitol, arabitol, glycerol and glycol, di- and.¾ligosaccharides such as lactose and maltose, polysaccharides such as cellulose, starch, xylan and mannan, complex industrial residues such as corn steep liquor, milk whey, soy bran, soybean hulls, and distiller's spent grains and different biomass residues. _;

The carbon source used for enzyme production is such that it comprises primarily biomass hydrolysate. When describing that the carbon source "comprises primarily", it is meant that≥ 50 wt% of the carbon source comprises biomass hydrolysate. When describing biomass hydrolysate as a carbon source, all contained-mono- and oligomeric forms of the sugars glucose, xylose, arabinose, mannose, galactose and rhamnose contained are considered. As is known to those skilled in the art, such sugars may be quantified from a sample by hydrolyzing all sugars into monomeric form with an acid such as trifluoroacetic acid (TFA) or sulphuric acid and detecting the sugars using high-performance liquid chromatography (HPLC). The growth medium may or may not be additionally supplemented with other carbon sources in addition to those previously mentioned.

Nitrogen sources suitable for enzyme production include but are not limited to peptone, yeast extract, whey protein, urea, ammonia and ammonium salts. Suitable sources of phosphorus include salts of phosphate, such as potassium phosphate and sodium phosphate. Suitable other elements to be used in enzyme production include potassium, calcium, chloride, magnesium, manganese, km, zinc, copper and cobalt.

The pH during enzyme production may be maintained within the desired range through the addition of substance that is an acid or a base. Preferred acids include phosphoric acid, sulfuric acid and hydrochloric acid. Preferred bases include ammonia, potassium hydroxide and sodium hydroxide. Oxygen may be added to the growtfi medium during cultivation by feeding compressed air, oxygen or a mixture of oxygen with other gasses. The cultivation medium may be agitated using impellers, gas flows, or any other suitable means.

During enzyme production, the fungus may produce enzymes within its cells (intracellular enzymes) or secrete them into the external medium (extracellular enzymes) or it may do both. The presence of enzymes may be evaluated by measuring protein concentrations from the extracellular medium. Protein may be measured by methods such as those described by Lowry (28) and Bradford (29). As is known to those skilled in the art, values measured for protein concentration vary widely depending on the methodology employed. Therefore, when referring to "enzyme

concentration", here a value is meant that is obtained by the following method: A culture supernatant sample is diluted with 50-millimolar pH 5.0 sodium-citrate buffer so that the final extracellular protein concentration is within the range 0.3 - 1.5 g/L. Proteins from t ¾ sample are precipitated by adding four volumes of ice-cold acetone and maintaining the sample for one hour at -20°C. The proteins are pelleted by centrifuging for 5 minutes at 20.000 g and 4°C, the supernatant removed and the protein pellet allowed to dry for 5 minutes at room temperature. The protein pellet is then resuspended in the original volume of buffer and the concentration measured using the method of Lowry (28) using a bovine serum albumin (BSA) standard.

The presence of suitable enzymes in the medium may also be studied using various methods for quantifying enzymatic activities. Such methods may be based on contacting a sample with a substrate, and measuring products released from the substrate. Suitable substrates are well known to those skilled in the art and include Avicel, carboxymethyl cellulose, hydroxyethyl cellulose, beechwood fylan, filter paper, cellobiose, 4-nitrophenyl-p-D-glucopyranoside, 4-nitrophenyl- -D-xylopyranoside, 4- nitrophenyl-a-L-arabinofuranoside and 4-methylumbelliferyj-P-D- lactopyranoside. The released products may be reducing sugars, 4- nitrophenol, 4-methylumbel!iferone or other compounds. Methods of quantification include but are not limited to the detection of reducing sugars using the DNS method (31 ), spectrophotometric detection of 4- nitrophenol using a wavelength of about 400 nm and fluorometric detection of 4-methylumbelliferone using an exciting wavelength of about 365 nm and detecting wavelength of about 445 nm.

During enzyme production, samples maybe withdrawn from the vessel. The samples can be analyzed by suitable means to determine the concentration of fungal cells, the concentration of proteins and the activities of enzymes. The determined parameters may be used to define the appropriate time to proceed to the second step of the process, hydrolysis.

Hydrolysis

Step b) of the disclosed process is know as "hydrolysis", and is considered to begin with the addition into the reactor of biomass substrate, that is optionally pre-treated. ^

The biomass substrate may be derived from a hardwood, a softwood or a grass. Preferred, non-limiting examples of biomass substrates include sugarcane bagasse, sugarcane straw, corn stover, wheat straw, barley straw, rye straw, rice straw, sorghum straw, wood chips, sawdust, switchgrass (Panicum virgatum), Giant Cane (Arundo donax), silvergrass (Miscanthus spp.) and empty palm fruit bunches.

With "pre-treatment" is meant a physical, thermal, chemical or other type of process that modifies the structure of biomass in a way that it becomes more susceptible to enzymatic degradation. Such susceptibility to enzymatic degradation can be evaluated, for example, by using an equal dose of the same enzyme for an equal amount of the biomass, for an equal amount of time under equal conditions of temperature, pH and agitation. Under such equal conditions, a greater amount of soluble sugars will be released from the biomass that is "more susceptible to enzymatic degradation".

Non-limiting examples of pre-treatments making biomass more susceptible to enzymatic degradation include hydrothermal pre-treatment, dilute-acid pre-treatment, steam-explosion, alkaline oxidation, the use of organic solvents and the use of ionic liquids. The pre-treatment may also comprise several different steps, including but not limited to those previously mentioned. The pre-treatment may modify the physical structure of the biomass and may or may not also modify the chemical composition of the biomass. The pre-treatment may or may not also convert part of the insoluble biomass into soluble form.

"Pre-treated biomass" as used herein describes a biomass that has passed through a process of pre-treatment and thus become more susceptible to enzymatic degradation than it was before the pre-treatment. The biomass may be a solid or a mixture of solids and liquids. The pre- treated biomass may undergo further processing such as washing, de- sizing or drying before being fed into the vesseJ.

The heat of the pre-treated biomass may be used to heat the contents of the vessel to the desired temperature. A desired hydrolysis temperature may be maintained in the vessel <using electric heater resistors or by circulating warm water around the vessel. The vessel may be cooled by circulating cool water around the vessel.

After the step of hydrolysis the soluble sugars may be separated from the insoluble solid residues of the process. Solid residues may include lignin, residual fibers and fungal cells. Methods that may be used for separating solids from liquids include but ave not limited to decantation, centrifugation, membrane filtration and the use of rotary drums.

With describing that the genetically engineered fungus is prevented from consuming "the major part of soluble sugars", it is meant that the fungus consumes no more than 50 wt% of the'soluble sugars released by the enzymes from the pre-treated biomass. N©n-limiting examples of conditions and mechanisms that may thus present the fungus include an increase in temperature, a decrease in soluble oxygen and the presence of one or more chemical inhibitors. Chemical inhibitors may be present in the pre-treated biomass or they may be produced by another organism or other organisms within the vessel. Suitable chemical inhibitors may. include furfural, hydroxymethylfurfural, organic acids, phenolic compounds and alcohols.

The soluble sugars produced in hydrolysis may be recovered or they may be fermented using another organism or other organisms. With "fermentation" here is meant a process by which an organism consumes the soluble sugars and produces a compound of interest. Such

compounds can be but are not limited to ethanol, butanol, propanol, acetic acid, lactic acid, farnesene, itaconic acid, adip?c acid, xylonic acid and glycolic acid. In a preferred embodiment, the desired compound is ethanol. In one embodiment, the desired end product may also be cell mass or a fraction of cell mass of the fermenting organism. The fermentation can be aerobic, meaning that the fermentation is carried out in the presence of oxygen, or anaerobic, meaning that it takes place in the absence of oxygen.

With "another organism or other organisms" is meant any organism belonging to a species other than that of the genetically engineered fungus used for "enzyme production". Another microorganism or other

microorganisms may be a bacterium, an archaeon, an alga, a yeast or a fungus. In preferred embodiments of this invention, the other organism is Saccharomyces spp., a Pichia spp., a Candida spp., a Zymomonas spp., an Eschericia spp., a Bacillus spp., a Lactobacillus spp., a Lactococcus spp., a Pseudomonas spp., an Alcaligenes sp ., a Clostridium spp., an Aspergillus spp. In a most preferred embodiment of this invention, the fermenting organism is the yeast Saccharomyces cerevisiae.

"Fermentation" as used herein shall not fee confused with the first step of the process, which comprises fermentation with a genetically engineered fungus, but which is herein referred to as "enzyme production".

The soluble sugars may be fermented wii:h another organism or other organisms after a filtration step or without prior filtration of the bydrolysate. In one embodiment, the fermentation of the soluble sugars may take place after the hydrolysis step (separate hydrolysis and fermentation - SHF). In another embodiment, the fermentation of the soluble sugars is

simultaneous with the hydrolysis step (simultaneous saccharification and fermentation - SSF). In yet another embodiment, the fermentation of the soluble sugars using another organism or other organisms commences after only a part of the hydrolysis step has been completed (hybrid hydrolysis and fermentation - HHF). In the case of an SSF process, a separate smaller hydrolysis tank may be used with a fraction of pre-treated biomass and a fraction of the enzyme produced in the "enzyme

production" step of the process, to generate a suitable sugar stream for a new round of "enzyme production".

With "separate hydrolysis and fermentation (SHF)", is meant a process where the steps of hydrolysis and subsequent fermentation using another organism or other organisms are temporally and spatially delimited. The steps of hydrolysis and fermentation may be performed at different temperatures and at different pH. In one preferred embodiment of the invention, the hydrolysis step is performed at a temperature of about 40°C to about 50°C, for a period of about 12 hours to about 120 hours, and a pH of about 4.5 to about 5.5. In this preferred embodiment, the genetically modified fungus may be prevented from consuming soluble sugars by the elevated temperature. A fermentation step with another organism or another organism may then be carried out with or without prior filtering the hydrolysate at.a temperature of about 25°C to about 35°C, for a period of about 12 hours to about 120 hours. In a preferred embodiment, the other organism is S. cerevisiae, and the fermentation product is ethanol.

When referring to "simultaneous saccharification and fermentation (SSF)",- a process is meant where enzymatic hydrolysis of the pre-treated biomass and fermentation of the released soliible sugars by another organism or other organisms take place simultaneously. In one preferred embodiment of the invention, such a process takes place at a temperature of about 25°C to about 35°C, at a pH of about 4.0 to about 6.0, for a period of about 10 hours to about 240 hours. In a preferred embodiment, the fermenting organism is S. cerevisiae and the fermentation product is ethanol. In this preferred embodiment, the genetically modified fungus may be prevented from consuming soluble sugars due to the accumulation of ethanol, which may be toxic to the fungus.

A process described as "hybrid hydrolysis and fermentation (HHF)" combines features of the SHF and SSF processes. In one preferred embodiment of this invention, the hydrolysis step may be shorter than in the SHF process. The hydrolysis may take from about 4 hours to about 48 hours or any duration there bet een. The temperature may then be lowered to from about 20°C to about 40°C and another organism or other organisms added to the vessel. The fermentation of the soluble sugars may then take place while the enzymes still actively degrade the pre- treated biomass. In one preferred embodiment, part of the hydrolysate is withdrawn from the vessel before adding another organism or other organisms and this hydrolysate fraction used "enzyme production" in another vessel or in the same vessel at a later point in time.

In the most preferred embodiment, the process is an HHF-type process.

The other organism or other organisms may be added into the vessel in the form of spores or as a cell-inoculum. The methods and the vessels used to produce the inoculum of the other organism or other organisms used for "fermentation" may be the same as those used to produce the inoculum of the genetically engineered fungus used in "enzyme

production", or they may be different.

Examples

Example 1 - Genetic engineering of strains

Example 1.1 - Creation of strain VTT-BR-C0019

The plasmid pVTTBR43 (Figure 2A) was created using standard molecular cloning techniques and E. coli as the cloning host. The plasmid spans 10,553 base pairs (bp) and contains the following elements: ColE origin of replication and KanR kanamycin resistance marker for

propagation of the plasmid within E. coli. Further, it contains the T. reesei Xyr1 transcription factor under the control of the T. reesei pyruvate decarboxylase promoter (Ppdc) and terminator (Tpdc). The Xyr1 coding sequence contains the mutations G2461T and A2463C relative to the native sequence found in T. reesei genomic D*JA, resulting in the substitution of a valine residue at position 821 for a phenylalanine residue in the protein product of the gene. The plasmid contains the hygromycin phosphotransferase gene from Streptomyces hygroscopius under the control of the gdpA promoter (PgdpA) and the irpC terminator (TtrpC) from Aspergillus nidulans. It also contains the acetamidase (amdS) gene from A. nidulans which could be used for negative selection. Both markers (hph and amdS) lie between loxP sites, allowing marker removal using a Cre- recombinase. The plasmid was digested with the restriction enzyme Mssl (Thermo Scientific) and the resulting 10,334 kb fragment was isolated from an agarose electrophoresis gel using a commercial kit (Zymo Research). Around 5 pg of the linear DNA was used in PEG mediated transformation of protoplasts of T. reesei VTT-BR-C0001 essentially as described in (25). The protoplasts were plated in top-agar containing 50 pg/mL hygromycin B (Calbiochem) and allowed to grow for 5-7 days before isolation of individual colonies. Screening transformant coionies for increased extracellular enzyme production on glucose in shakeflask culture lead to the identification of VTT-BR-C00 9, which produces significantly more extracellular enzymes than the parental strain VTT-BR-C0001 (Figure 3A). Integration of the transformation cassette was verified using PCR.

Example 1.2 - Creation of strain VTT-BR-C0020

The plasmid pVTTBR54 (Figure 2B) was created using standard molecular cloning techniques and E. coli as the cloning host. The plasmid spans 10,994 bp and contains the following elements: ColE origin of replication and KanR kanamycin resistance marker for propagation of the plasmid within E. coli. Further, it contains the ? ^" . ^' emersonii Cel3A beta- glucosidase under the control of the T. reesei xyn11 B xylanase promoter (Pxyn11 B) and terminator (Txyn 11 B). The plasmid contains the

phosphinothricin acetyltransferase gene from Streptomyces hygroscopius under the control of the gdpA promoter (PgdpA) and the trpC terminator (TtrpC) from A. nidulans. The plasmid was digested with the restriction enzymes Mssl and Nhel (Thermo Scientific) and the resulting 7,703 kb fragment was isolated from an agarose electrophoresis gel using a commercial kit (Zymo Research). Around 5 pg of the linear DNA was used in PEG mediated transformation of protoplasts of T. reese/ VTT-BR-C0019 as previously described. The protoplasts were plated in top-agar containing 1000 pg/mL glufosinate-ammonium (Sigma) and allowed to grow for 5-7 days before isolation of individual colonies. Screening transformant colonies for increased β-glucosid ^' ase secretion in shakeflask culture lead to the identification of VTT-BR-C0020, which produces significantly more β-glucosidase than the parental strain VTT-BR-C0019 (Figure 3B). Integration of the transformation cassette was again verified using PCR.

Example 2 - Enzyme production by strains VTT-B -C0001, - C0019 and C0020 grown on lactose and glucose as primary carbon sources.

Shakeflask cultivations were performed to study enzyme production by strains VTT-BR-C0001 , -CO019 and -COO2 on an inducing carbon source comprising Avicel microcrystaliine cellulose and lactose, and alternatively on the repressing carbon source glucose. Cultures of 50 m!_ were grown in triplicate 250 mL Erlenmeyer flasks containing a basal medium with the following composition: 3 g/L v-east extract, 5 g/L

(NH ₄ ) ₂ S0 ₄ , 15 g/L KH ₂ P0 ₄ , 0.6 g/L Mg ₂ S0 ₄ · 7H ₂ 0, 0.45 g/L CaCI ₂ , 5 mg/L FeS0 ^■ 7H ₂ 0, 2 mg/L CoCI ₂ · 7H ₂ 0, 1.6 mg/L ¾lnS0 ₄ · 4H ₂ 0, 1.4 mg/L ZnS0 ₄ · 7H ₂ 0 and 50 mM citric acid. The pH of the flasks was adjusted to 4.8 with KOH and the media autoclaved. Sterile lactose or glucose solution was added to a final concentration of 50 g/L and the flasks inoculated with ~ 10 ⁷ spores of the relevant strain. The cultivations were allowed to proceed for seven days at 28°C and with 200 rpm agitation, with aseptic sampling on days 3, 5 and, 7. The samples were centrifuged and the supernatants frozen for subsequent analysis. For quantification of extracellular protein concentrations (Figure 3A) the samples were first diluted in 50 mM Na-citrate buffer pH 5.0, precipitated with 4 volumes of acetone for one hour at -20°C. The proteins were pelleted at 4°C 14,000g in a tabletop centrifuge and resuspended in the original volume of buffer. The protein concentration was then quantified Rising the BioRad DC II kit, using bovine serum. albumin (BSA) as standard, β-glucosidase activities were measured from the supernatants using the substrate nitrophenyl-β- D-glucopyranoside. Released 4-nitrophenol wis quantified using a plate spectrophotometer at 405 nm and converted to enzymatic activity using a standard curve. One unit is defined as the amount of enzyme releasing pmol of 4-nitrophenol per minute. The results-show that the genetic modifications altered total enzyme production by strains VTT-BR-C0019 and -C0020 compared to the parental strain VTT-BR-C0001 relatively little when the strains were grown on an inducing Avicel-lactose medium

(Figure 3A). However, on glucose these strains produced markedly more enzyme than the parental strain, a phenotype conferred by the

Xyr1_V821F overexpression in VTT-BR-C001S and maintained in VTT- BR-C0020. In addition, strain VTT-BR-C0020 ¾an be seen to produce high levels of β-glucosidase on both media (Figure 3B).

Example 3 - Profile of enzymes secreted by modified strains VTT-BR-C0001, -C001S and C0020 grown oil? lactose and glucose as primary carbon sources.

The final cultivation samples (day 7) frorrr Example 2 were subjected to various enzymatic assays to determine the relative proportion of different lignocellulolytic enzymes produced by the strains (Figure 4). The activities measured were: xylanase, endoglucanase, β-g!ucosidase and β- xylosidase and total cellulase (FPase). All reactions were carried out at 50°C in 50 mM Na-citrate pH 5.0 buffer for 5 to-10 minutes (60 minutes for FPase). The substrates used for xylanase and endoglucanase

measurements were beechwood xylan and carboxymethylcelluiose, respectively. The released sugars were quantified using the dinitrosalisylic acid (DNS) method and the results converted into enzymatic activities (U/mL) using xylose or glucose as standard as appropriate, β-glucosidase and β-xylosidase activities were measured using a nitrophenyi^-D- glucopyranoside and 4-nitrophenyl^-D-xylopyranoside substrate, respectively. Reactions were terminated with G.5 volumes of 0.5 M

Na2C0 ₃ and the released 4-nitrophenc! determined using a

spectrophotometer at 405 nm wavelength. The result was converted into enzymatic activity using a standard curve of 4¾iitrophenol. Activity towards filter paper was measured using the standard method (31 ). in all cases one unit is defined as the amount of enzyme releasing one pmol of reducing sugar or 4-nitrophenol per minute under the stated conditions. Figure 4 displays the obtained values for specific activities (U/mg) in the numeric labels, while the bars show the results as a fraction of the highest obtained result for each activity. The results show a relative increase of xylanolytic enzymes in the secretome of VTT- R-C0019, while the increase in β-glucosidase activity in VTT-BR-C0020 is clearly evident.

Example 4 - Hydrolysis of hydrothermally pre-treated sugarcane bagasse using the enzymes secreted by strains VTT-BR-C0001, - C0019 and -C0020

The final cultivation supernatants from Example 2 were used to hydrolyze washed hydrothermally pre-treated*5ugarcane bagasse to evaluate the relative performance of each enzyme mixture. Hydrolysis reactions of a total weight of 1 gram were carried out in 2 mi_ Eppendorf tubes using an Inteilimixer apparatus (Elmi) and the program 2u 18 rpm for agitation. The apparatus was maintained inside an incubation chamber with the temperature set to 45°C. Based on-ackJ hydrolysis and HPLC quantification the substrate presented a glucan and xylan content of 40.7 % and 4.0 % of dry weight, respectively. 100 mg dry substrate was weighed into each tube. The reactions were completed with buffer (50 mM Na-citrate pH 5.0) and enzyme for a final enzyme loading of 10 mg/g dry substrate and a solids content of 10 wt%. Hydrolysis reactions were conducted for 24, 48 and 72 hours, the entire contents of each tube diluted with 9 mL distilled water and the released reducing sugars quantified using the DNS method. Additionally, the sugars glucose, xylose and cellobiose were quantified from the final (72h) hydrolysis samples. For each enzyme and time-point, quadruplicate tubes were prepared and used to calculate standard deviations. The enzymes produced by strain -VTT-BR-C0001 on glucose were excluded from this experiment due to their low

concentration. Figure 5A shows the reducing sugars released from the biomass as a function of time as measured by DNS. The results

demonstrate that the enzymes produced by strain VTT-BR-C0019 on the inducing Avicel-lactose medium (Filled squares) had a slightly decreased performance compared to the enzymes produced by the parental strain VTT-BR-C0001 (Filled circles). The enzymes produced by strain VTT-BR- C0020 on the same medium (Filled triangles) had a dramatically improved performance due to the higher amount of β-glucosidase. The enzymes produced by VTT-BR-C00 9 (open squares) and -C0020 (open triangles) on glucose display an inferior performance compared to the enzymes produced on Avicel-lactose. However, the enzymes produced by VTT-BR- G0020 on glucose showed a comparable performance to the enzymes produced by the original strain VTT-BR-COOOI on Avicel-lactose. Figure 58 shows the glucose, xylose and cellobiose concentrations from the final hydrolysis samples. The results clearly show the importance of the additional beta-glucosidase activity in the enzymes produced by VTT-BR- C0020. Virtually no cellobiose accumulated when the bagasse was hydrolyzed with the enzymes from this strain. Example 5 - Use of strain VTT-BR-C0020. with a repressing industrial carbon source in a combined enzyme production and hydrolysis experiment

T. reesei strain VTT-BR-C0020 was used in a laboratory scale bioreactor experiment to demonstrate its utility in a single-vessel enzyme production and hydrolysis set-up. The results are summarized in Figure 6. In a first step, a BioFlo Celligen 115 bioreactor- (Eppendorf) was loaded with 750 mL of a medium containing: 5 g (NH ₄ )2S04, 15 g KH ₂ PO ₄ , 0.6 g Mg ₂ S0 ₄ · 7H ₂ 0, 0.45 g CaCI ₂ , 5 mg FeS0 ₄ · 7H ₂ 0, 2 mg CoCI ₂ · 7H ₂ 0, 1.6 mg MnS0 ₄ · 4H ₂ 0, 1.4 mg ZnS0 ₄ · 7H ₂ 0, 1 mL antifoam (J647 - Struktol) and sterilized by autoclaving. The carbon source used in the experiment was acid-inverted sugarcane molasses. This solution was diluted to a concentration of 200 g/L total sugars (glucose and fructose) with sterile water and 150 mL of this solution added to the reactor for a final sugar concentration of 3 % (w/v) at the beginning oHhe cultivation. To

commence enzyme production, 100 mL inoculum containing 3 g/L dry- weight of cells of the strain VTT-BR-C0020 grown in the same basai salt medium supplemented with 2% yeast extract and 3% inverted molasses sugar was added to the reactor: Enzyme production was allowed to proceed for 48 hours, maintaining temperature at 28°C, pH between 4.0 and 5.0 using 10 % ammonia and 2 M phosphoric acid. Compressed air was fed into the reactor at 0.7 SLPM. Samples were withdrawn at intervals and residual sugars, dry-weight, extracellular protein and β-glucosidase quantified (see Example 3 for measurements). After 48 hours, with the extracellular protein concentration reaching 5.3 g/L, the reactor was transferred to a laminar flow cabinet and 298 ! g of hydrothermally pre- treated bagasse (see Example 4) was added for a final bagasse

concentration of 10 wt%. The reactor was remounted and temperature increased to 45°C, gas flow into the reactor stopped and agitation set to 600 rpm. After 6 hours, 552.5 g of the same bagasse was added to the reactor for a final substrate concentration of 20 wt% in the hydrolysis step and agitation increased to 800 rpm. Hydrolysis was then allowed to proceed for a total of 96 hours (counting from t e first addition of bagasse). Samples were withdrawn at intervals, diluted 10-fold with distilled water and released sugars quantified using DNS and HPLC. The final broth after 96 hours was sterile filtered for use in Example 6.

Example 6 - Use of strain VTT-BR-C0G20 in a combined enzyme production, hydrolysis and ethanol fermentation process, with biomass hydrolysate as carbon source for Enzyme production

A further experiment was performed in a laboratory scale bioreactor to explore the ability of strain VTT-BR-C0020 to produce enzymes on hydrolysate sugars and its utility in the conversion of pre-treated biomass into soluble sugars and finally ethanol. The obtained results are

summarized m Figure 7. The experiment was set up to correspond to the preferred embodiment illustrated in Figure 1 A. Inoculum and reactor preparation were performed basically as descilbed in Example 5, albeit with a slightly lower working volume. The carbon source used was a biomass hydrolysate resulting from a single-vessel enzyme production and hydrolysis experiment similar to that described in Example 5. This hydrolysate had a sugars concentration of 60.3 g/L as measured by DNS using a glucose standard. After sterile filtration; 425 mL of this hydrolysate was added to 345 mL of salt solution prior to adding a 80mL fungal inoculum, so that the final concentration of suo'ar was 3% and the concentration of salts were equal to those used in Example 5. The dry weight of cells in the inocuium was measured Ό be 15 g/L Enzyme production was carried out for 52 hours, while Withdrawing samples to quantify dry-weight, extracellular protein, activty against pWPG and residual sugars.

After 52-hours of enzyme production, wis' j extracellular.protejn reaching 5,5 g/L, pH was corrected to 5.0 and-362.3 grams of pre-treated bagasse was aseptically added to the reactor for a solids loading of ~13 wt%. The temperature was then raised to 45° for hydrolysis and gas flow to the reactor stopped as previously (see Example 5). Another 362.3 grams of pre-treated bagasse was added after 6 hours, for a final solids loading of 20 wt%. Hydrolysis was allowed to proceed at 45°C for around 72 hours counting from the addition of the first portion of bagasse.

Samples were withdrawn at regular intervals, diluted with 10-fold with distilled water and released sugars quantified using HPLC. After 72 hours, the temperature was lowered to 33°C and a 100 mL inoculum of S.

cerevisae with a dry cell concentration of 7.5 g/L was added. Fermentation was then allowed to proceed for around 72 hours. The reactor was regularly sampled, and the samples used for the quantification of glucose, xylose and ethanol using HPLC. The results indicate that it is indeed possible to produce enzymes on biomass hydrolysate, hydrolyze biomass and ferment the released sugars into ethanol in sequential steps within the same reaction vessel.

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