MXPA00008786A - Genetic constructs and genetically modified microbes for enhanced production of beta-glucosidase - Google Patents

Abstract

This invention relates to the genetic modification of a microbe to enhance its production of an enzyme, beta-glucosidase, that is important in the cellulose conversion process. The inventors have discovered genetic constructs that, when expressed in recombinant microbes, dramatically increase the amount of beta-glucosidase produced relative to untransformed microbes. The genetic constructs comprise a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region. The increased level of beta-glucosidase significantly increases the efficiency of hydrolysis of cellulose to glucose by celulase enzymes, thereby enhancing the production of fuel ethanol from cellulose.

Description

GENETIC CONSTRUCTIONS AND GENETICALLY MODIFIED MICROBES FOR THE IMPROVED PRODUCTION OF BETA-GLUCOSIDASE BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to the genetic modification of microbes to improve the production of a commercially important enzyme, beta-glucosidase. This invention also relates to genetic constructs that dramatically increase the amount of beta-glucosidase produced by the microbes that contain these constructs. 2. BACKGROUND OF RELATED ART The possibility of producing ethanol from cellulose has received much attention due to the availability of large quantities of raw material, the desirability of avoiding the burning or sanitary filling of materials, and the cleaning of ethanol fuel. The advantages of this process for society are described in the cover story of the Atlantic Monthly, April 1996. The natural cellulose feeds for this process are referred to as "biomasses". Many types of biomass have been considered as food for the production of ethanol, including wood, agricultural residues, arable crops and municipal solid waste. These materials consist mainly of cellulose, hemicellulose and lignin. This invention can be applied to the conversion of cellulose to ethanol. Cellulose is a polymer of simple sugar, glucose, connected by beta-1,4 bonds. Cellulose is very resistant to degradation or depolymerization by acids, enzymes or microorganisms. Once the cellulose is converted to glucose, the resulting sugar is easily fermented to ethanol using yeast. The difficult challenge of the process is to convert ceLulose to glucose. The oldest methods studied to convert cellulose to glucose are based on acid hydrolysis (reviewed by Grethlein, "Chemical Breakdown of Cellulosic Materials," J. Appl. Chem. Biotechnol. 28: 296-308 (1978)). This process may include the use of concentrated or diluted acids. The process with concentrated acids produces a high yield of glucose, but the recovery of the acid, the specialized construction materials required, and the need to minimize the water in the system are serious disadvantages of this process. The process with dilute acids uses low levels of acid to overcome the need for chemical recovery. However, the maximum glucose yield is only about 55% of the cellulose, and a high degree of degradation product production can inhibit fermentation to ethanol by the yeast. These problems have prevented the acid hydrolysis process from reaching commercialization. To overcome the problems of the acid hydrolysis process, cellulose conversion processes have been focused more recently on enzymatic hydrolysis, using cellulase enzymes. The enzymatic hydrolysis of cellulose is carried out by mixing the substrate and water to obtain a slurry of 5% to 12% cellulose and adding 5 to 50 international units (IU) of cellulase enzymes per cellulose gm. Typically, hydrolysis runs for 12 to 150 hours at 35-60 ° C, pH 4-6 .. Many microbes make enzymes that hydrolyze cellulose, including the wood decay fungus Tri choderma, the compost bacteria Thermomonospora, Ba cil l us, and Cel l ul omonas; Streptomyces; and the mushrooms Humi cola, Aspergi l l us and Fusa ri um. The enzymes elaborated by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose to glucose: endoglucanases (EG), cellobiohydrolases (CBH), and beta-glucosidase. The enzymes of EG and CBH are collectively referred to as "cellulose". EG enzymes cut the cellulose polymer at random locations, opening it until attacked by the CBH enzymes. As an example, Tri choderma strains produce at least four enzymes other than EG, known as EGI, EGII, EGIII and EGV. The CBH enzymes sequentially release cellobiose molecules from the ends of the cellulose polymer. The cellobiose is the glucose dimer beta-1, 4-water soluble bond. There are two main CBH enzymes produced by Tri choderma, CBHI and CBHII.

Beta-glucosidase enzymes hydrolyze cellobiose to glucose. Tri ch oderma produces a beta-glucosidase enzyme. This final step in the hydrolysis of cellulose that is catalyzed by beta-glucosidase is important, because glucose is easily fermented to ethanol by a variety of yeasts while cellobiose does not. Any cellobiose that remains at the end of the hydrolysis represents a loss of ethanol production. More importantly, cellobiose is an extremely potent inhibitor of the CBH and EG enzymes. The cellobiose decreases the rate of hydrolysis of the CBH and EG enzymes of Tri choderma by 50% at a concentration of only 3.3 g / L. The decrease in the rate of hydrolysis necessitates the addition of higher levels of cellulase enzymes, which have an adverse impact on the overall economy of the process. Therefore, the accumulation of cellobiose during hydrolysis is extremely undesirable for the production of ethanol. The accumulation of cellobiose has been a major problem in enzymatic hydrolysis because Tri choderma and other microbes that produce cellulose make very little beta-glucosidase. Less than 1% of the total protein made by Tri choderma is beta-glucosidase. The low amount of beta-glucosidase results in a deficit in the ability to hydrolyze cellobiose to glucose and an accumulation of 10 to 20 g / L of cellobiose during hydrolysis. This high level of cellobiose increases the amount of cellulase required by 10 times with respect to that if an adequate amount of beta-glucosidase is present. Several approaches have been proposed to overcome this deficit of beta-glucosidase in cellulase enzymes. One approach has been to produce beta-glucosidase using microbes that produce little cellulase, and to add this beta-glucosidase exogenously to the cellulase enzyme to improve hydrolysis. The most successful of these microbes that produce beta-glucosidase has been Aspergill us niger and Aspergi ll us phoeni c ± s. The beta-glucosidase from these microbes is commercially available as Novozym 188 from Novo Nordisk. However, the quantities required are much more expensive for a commercial biomass for the ethanol operation.

A second approach to overcome the deficit of beta-glucosidase is to carry out cellulose hydrolysis - simultaneously with fermentation of glucose by yeast, the so-called simultaneous process of saccharification and fermentation (SSF). In an SSF system, the glucose fermentation removes it from the solution. Glucose is a • potent inhibitor of beta-glucosidase, so that SSF is an attempt to increase the efficiency of beta-glucosidase. However, SSF systems are not yet commercially available because the operating temperature for the yeast of 28 ° C is too low for the 50 ° C conditions required by the cellulase; the operation at an engagement temperature of 37 ° C is inefficient and prone to microbial contamination. A third approach to overcome the deficit of beta-glucosidase is to use genetic management to overexpress the enzyme and increase its production by Tri choderma. This approach was taken by Barnett, Berka, and Fowler, in "Cloning and Amplification of the Gene Encoding an Extracellular ß-glucosidase from Tri Choderma reesei: Evidence for Improved Rates of Saccharification of Cellulosic Substrates", Bio / Technology, Volume 9, June of 1991, page 562-567, in the same is referred to as "Barnett, et al.,"; and Fowler, Barnett and Shoemaker in WO 92/10581, "Improved Saccharification of Cellulose by Cloning and Amplification of the β-glucosidase gene of Tri choderma reesei", it is referred to as "Fowler, et al.". Both Barnett et al., And Fowler et al., Describe the insertion of multiple copies of the beta-glucosidase gene in the P40 strain of Tri choderma rees ei. Both groups constructed the plasmid pSASß-glu, a transformation vector containing the genomic beta-glucosidase gene of T. re esei and the amdS selectable marker. The a dS gene is from Aspergi l l us nidulans and codes for the enzyme acetamidase, which allows transformed cells to grow in acetamide as a single source of nitrogen. The T reesei does not contain a functional equivalent to the amdS gene and is therefore unable to use acetamide as a nitrogen source. The transformed cells contained 10 to 15 copies of the beta-glucosidase gene and produced 5.5 times more beta-glucosidase "than the untransformed cells. The improved production of beta-glucosidase obtained by Barnett et al. and Fowler et al., is not sufficient to mitigate the beta-glucosidase deficit for cellulose hydrolysis. The amount of beta-glucosidase produced by the natural strains of Tri choderma, for example, must be increased at least 10 times to meet the requirements of cellulose hydrolysis. When proteins are overexpressed in Tri choderma, one strategy is to link the gene of interest directly to the cbhl promoter or to the cbhl secretion signal. Since cbhl is the most abundant protein produced by Tri choderma under cellulase induction conditions, the promoter and secretion signal cbh l are thought to be very effective in the direction of transcription and secretion of proteins encoded by a gene placed after of them in a genetic construction. This strategy has been used successfully to overexpress Tri choderma proteins and other microorganisms (Margoller-Clark, Hayes, Harman and Penttila, 1996, "? Mproved Production of Tri-choderma ha rzían um endoch.it inase by expression in Tri choderma reesei", Appl. Environ, Microbiol. 62 (6): 2145-2151; Joutsjouki, Torkkeli and Nevalainen, 1993, "Transformation of Tri-chorema reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Tri choderma reesei", Curr. Genet 24: 223-228; Karhunen, Mantyla, Nevalainen and Souminen, 1993, "High Frequency One-Step Gene Replacement in Tri Choderma Reesei 1. Endoglucanase I overproduction", Mol. Gen. Genet. 241: 515-522). Despite a significant amount of search effort, there has not been a means to sufficiently produce high levels of beta-glucosidase. This process would be a big step towards producing fuel alcohol from cellulose.

BRIEF DESCRIPTION OF THE INVENTION The inventors have made a discovery that allows the production of the beta-glucosidase enzyme levels higher than those currently available. The high levels of beta-glucosidase improve the efficiency of the enzymatic hydrolysis of cellulose to glucose. The resulting decrease in the enzyme requirement, the increased conversion to cellulose, decrease in hydrolysis time, or a combination of these advantages, decreases the total costs of the process to convert cellulose to ethanol. The inventors have discovered genetic constructs that significantly increase the production of beta-glucosidase by recombinant microbes in which the constructs are expressed. Genetic constructs that accomplish this task comprise DNA sequences that encode a mature beta-glucosidase enzyme and a xylanase secretion signal. As far as the inventors are aware, there are no previous reports that the binding of the xylanase secretion signal to the mature beta-glucosidase enzyme increases the production of glucosidase. Furthermore, the inventors are not aware of previous reports that the binding of the xylanase secretion signal to any mature non-xylanase protein increases the production of the protein. The inventors have discovered this surprising and unreported result. Additionally, it was surprising that the use of the xylanase secretion signal resulted in higher beta-glucosidase levels than the use of the cbh1 secretion signal. Since xylanase comprises a much smaller proportion of the total protein produced by Tri choderma than does cbhl (5% and 60%, respectively), it would be expected that the cbhl secretion signal would be more effective. The reasons why the binding of the xylanase secretion signal to the mature beta-glucoside enzyme increases the production of beta-glucosidase are not known, but may be related to the similarity in length between beta-secretion signals glucosidase and xylanase or the lower abundance of xylanase against which recombinant beta-glucosidase must compete for cell secretion. However, the practice of the invention is not limited by these or any other specific reason. The present invention is not anticipated by Barnett et al., And Fowler et al., Who each discovered improved expression of beta-glucosidase by recombinant means. The genetic construction of Barnett et al. and Fowler et al., comprises the beta-glucosidase promoter, the coding region and the secretion signal. The methods used by Barnett et al. and Fcwler et al, are not as effective as the methods taught by the inventors, and do not anticipate the genetic constructions of the present invention. In one aspect of the invention, a genetically modified microbe to produce beta-glucosidase comprises a beta-glucosidase construct not present in an untransformed microbe from which the genetically modified microbe is derived, this beta-glucosidase construct having a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region, wherein the genetically modified microbe is selected from the group consisting of Tri choderma, Humi cola, Fusari um, Strepizomyces, Thermo onospora j- Ba cill us r Cel l ul omona. s, and Aspergillus, and where the genetically modified microbe produces an approximately 10-fold increase in beta-glucosidase production relative to the untransformed microbe. In another aspect, the invention includes a genetic construction of beta-glucosidase comprising a promoter, a xylanase secretion signal, and a mature coding region of beta-glucosidase, wherein the genetic construct of beta-glucosidase, when it is introduced and expressed in the untransformed microbial host selected from the group consisting of Tri choderma, Humi cola, Fusari um, Streptomyces, Thermomonospora, Ba cillus, Cell ul omona s, and Aspergillus, produces at least about a 10-fold increase in beta-glucosidase production relative to the untransformed microbial host. In yet another aspect of the invention, a genetically modified Tri choderma microbe to produce beta-glucosidase comprises a beta-glucosidase construct not present in an untransformed Tri ch oderma microbe, the beta-glucosidase construct having a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region, wherein the genetically modified Tri ch oderma produces at least about a 10-fold increase in beta-glucosidase production relative to the Tri choderma microbe turned. In yet another aspect, the present invention includes a genetically modified Tri choderma reesei microbe * to produce beta-glucosidase comprising a beta-glucosidase construct not present in an untransformed Tri choderma reesei microbe, the beta-glucosidase construct that it has a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region, where the genetically modified Tri ch oderma rees microbe produces at least about a 10-fold increase in beta-glucosidase production. glucosidase in relation to the non-transformed microbe. In yet another aspect of the invention, a beta-glucosidase genetic construct comprises a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region, wherein the genetic construct of beta-glucosidase, when introduced in and expressed in a Tri Choderma microbe, produces at least approximately a 10-fold increase in beta-glucosidase production relative to the untransformed microbe from Tri-choderma. In yet a further aspect of the present invention, a genetic construct of beta-glucosidase comprises a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region, wherein the genetic construct of beta-glucosidase, when it is introduced and expressed in a microbe of Tri choderma reesei, producing at least about a 10-fold increase in the production of beta-glucosidase relative to an untransformed microbe of Tri ch oderma reesei. Other aspects of the invention will be better understood and the advantages thereof will be apparent in view of the following detailed description of the preferred embodiments and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Restriction map of the pCBGl-TV vector and the amino acid sequence of the secretion signal binding of cbhl signal / mature beta-glucosidase. Figure 2: Restriction map of the pXBGl-TV vector and the amino acid sequence of the xylanase II / beta-glucosidase secretion signal binding matures. Figure 3: Restriction map of the vector pC / XBG (Xbal) -TV and the amino acid sequence of the signal binding of xylanase II / beta-glucosidase secretion mature. Figure 4: Southern blot of genomic DNA isolated from strain RutC30 and M2C38 from T. reesei and probed with a labeled DNA fragment comprising the xylanase promoter M2C38 plus the secretion signal. Figure 5: Southern blot of genomic DNA isolated from strains RutC30 and M2C38 of T. rees ei and probed with a labeled DNA fragment comprising the mature beta-glucosidase coding region of M2C38.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Preferred embodiments of this invention are described by first defining the following terms: Beta-glucosidase is an enzyme that hydrolyses the glucose dimer, cellobiose to glucose. There are many microbes that produce beta-glucosidase, and the properties of these enzymes vary, including structure, molecular weight, three-dimensional orientation, amino acid composition and active site) and catalytic activity (velocity and kinetics of cellobiose hydrolysis, and ability to act on other substrates). Nevertheless, in all cases the beta-glucosidase enzyme. can hydrolyze cellobiose to glucose. This can also be referred to as a mature beta-glucosidase enzyme when the active enzyme does not contain a signal peptide of beta-glucosidase secretion. The beta-glucosidase preferred to practice the invention is the beta-glucosidase prepared by Tri ch oderma. This beta-glucosidase enzyme has a molecular weight of 74,000 (as measured by SDS-polyacrylamide gel electrophoresis) and has an isoelectric point of 8.3 (as measured by polyacrylamide gel electrophoresis with non-denaturing isoelectric focusing) The beta-glucosidase gene is a region of DNA that codes for the production of the beta-glucosidase enzyme All the microbes that produce beta-glucosidase have at least one beta-glucosidase gene A natural beta-glucosidase gene comprises a beta-glucosidase promoter, a secretion signal, a coding region and a transcriptional terminator Microbes that do not produce beta-glucosidase generally do not contain an active or functional beta-glucosidase gene. glucosidase is the DNA sequence that codes for the beta-glucosidase secretion signal peptide.The beta-glucosidase secretion signal peptide is the peptide sequence pr is present in the amino terminus of the beta-glucosidase-encoded enzyme that is subsequently removed during the export of the mature beta-glucosidase enzyme out of the microbial cells. The secretion signal may comprise a pro-peptide, a pre-peptide or both. The mature beta-glucosidase coding region comprises the DNA sequence necessary to encode the functional enzyme of beta-glucosidase, as isolated from the filtrates of extracellular cultures, but not the secretion signal of beta-glucosidase. Xylanase is an enzyme that hydrolyzes xylan to xylose. There are many microbes that make xylanase, and the properties of these enzymes vary, including structure (molecular weight, three-dimensional orientation, amino acid composition and active site) and catalytic activity (speed and kinetics of xylan hydrolysis and ability to act on other substrates However, in all cases the xylanase enzyme can hydrolyze xylan to xylose This can also be referred to as a mature xylanase enzyme when the active enzyme does not contain a signal peptide of xylanase secretion Some of the commercial xylanases more important are classified as xylanases from family 11. A xylanase enzyme is classified in family 11 if it possesses the amino acids common to family 11, including two glutamic acid residues that serve as the essential catalytic residues. 86 and 177 by the numbering of xylanase II of Tri ch oderma reesei. The xylanases from family 11 are described in Wakarchuck, et al., Protein Science 3: 467-475 (1994). - The xylanase gene is a region of DNA that codes for the production of xylanase enzyme. All microbes that produce xylanase have at least one xylanase gene. A natural xylanase gene comprises a xylanase promoter, a secretion signal, a coding region and a transcriptional terminator. Microbes that do not produce xylanase do not generally contain a functional or active xylanase gene. The xylanase secretion signal is the DNA sequence that codes for the xylanase secretion signal peptide.

The xylanase secretion signal peptide is the peptide sequence present in the amino terminus of the encoded xylanase enzyme that is subsequently removed during the export of the mature xylanase enzyme out of the microbial cells. The secretion signal may comprise a pro-peptide, a pre-peptide or both. Cellulase is an enzyme that hydrolyzes cellulose to short, bet-1, 4-linked glucose oligomers, including celotetraose, celotriose, and cellobiose. There are many microbes that make one or more cellulase enzymes frequently classified as cellobiohydrolases or endoglucanases. The properties of these enzymes vary, including structure (molecular weight, three-dimensional orientation, amino acid composition, and active site) and catalytic activity (speed and kinetics of xylan hydrolysis, and the ability to act on other substrates). However, in all cases the cellulase enzymes can hydrolyze cellulose to short beta-1,4-linked oligomers of glucose, including celotetraose, celotriose and cellobiose. The genetic construction of beta-glucosidase refers to a gene comprising the elements necessary to produce beta-glucosidase. These include: to. A coding region of mature beta-glucosidase. In a preferred embodiment, the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a Tri ch oderma gene. The DNA sequence of the mature beta-glucosidase coding region of Tri choderma reesei can be found in Figure 1 of Barnett, et al. Those skilled in the art are aware that a structural, natural region can be modified by replacement, substitution, addition or elimination of one or more nucleic acids without changing their function. The practice of the invention encompasses and is not restricted by these alterations to the mature beta-glucosidase coding region. b. A xylanase secretion signal In a preferred embodiment, the xylanase secretion signal comprises a xylanase secretion signal from a family of xylanase gene 11. In a more preferred embodiment, the xylanase gene from family 11 is a Xylanase gene from Tri Choderma. In yet a more preferred embodiment, the xylanase secretion signal comprises a xylanase secretion signal from the xylanase I gene of Tri choderma (xln l) or the xylanase II gene (xln2). The DNA sequences of the secretion signals xlnl and xln2 of Tri choderma can be found in Figures 3 and 2, respectively, by Torronen, Mach, Messner, Gonzalez, Kalkkinen, Harkki and Kubicek, "The two major xylasases from Trichoderma reesei : characterization of both enzymes and genes ", Bio / Technology in 10: 1461-1465, 1992 (the genetic identifications in the legends of the figures, as published, are inverted). Those skilled in the art are aware that a natural secretion signal can be modified by replacement, substitution, addition or elimination of one or more nucleic acids without changing their function. The practice of the invention encompasses and is not restricted by these alterations to the xylanase secretion signal. c. Promoter The practice of the invention is not restricted by the choice of the promoter in the genetic construction. However, the preferred promoters are the cbhl, cbh2, egl, egl, eg3, eg5, xlnl and xlnl promoters of Tri choderma. The cbhl DNA sequence of Tri choderma reesei is deposited in GenBank under accession number D86235. Those skilled in the art are aware that a natural promoter can be modified by replacement, substitution, addition or elimination of one or more nucleotides without changing its function. The practice of the invention encompasses and is not restricted by these alterations to the promoter. d. Additional sequences between the xylanase secretion signal and the mature beta-glucosidase coding region The genetic constructs described in Examples 5, 6 and 7 contain nine additional base pairs of the DNA sequence as shown in Figures 1- 3; the first three encode for the glutamine residue after the secretion signal of the xylanase II gene of Tri ch oderma rees ei and the remaining six result from the insertion and / or modification of the unique restriction sites used to bind the signal of secretion of xylanase to the coding region of mature beta-glucosidase. These DNA sequences result in the presence of additional amino acids between the xylanase secretion signal peptide and the mature beta-glucosidase enzyme. These DNA sequences, which may be natural or synthetic, may encode one or more of the amino acids of the mature xylanase protein corresponding to the xylanase secretion signal encoded by the construct or may result from the addition of enzyme sites of restriction necessary to bind the xylanase secretion signal peptide and the mature beta-glucosidase enzyme. The practice of the invention encompasses but is not limited by the presence of additional DNA sequences between the xylanase secretion signal and the mature beta-glucosidase coding region. and. Other elements The genetic construct contains a transcriptional terminator immediately in the 5 'direction of the mature beta-glucosidase coding region. The practice of the invention is not limited by the choice of the transcriptional terminator and can include this DNA in the 3 'direction (ie, at the 3' end) of the terminator codon of any known coding region since it is sufficient to direct the transcription termination by RNA polymerase. The transcriptional terminator present on the 3 'of the coding region of mature beta-glucosidase in the constructs described in Examples 5-7 comprises 1.9 kb of DNA 3' to the stop codon of the cbh2 gene Tri oderma ch. The DNA sequence of the first 553 base pairs of the cbh2 transcriptional terminator of Tri choderma reesei, which are located immediately in the 3 '(or 3') direction of the TAA terminator codon, can be found in Figure 2 of Chen, Gritzali and Stafford, "Nucleotide Sequence and Deduced Primary Structure of Cellobiohydrolase II from Tri chorema reesei", Bio / Technology 5: 274-278, 1987. The genetic construct contains a selectable marker that may be present at the 5 'direction or at the 3 'of the genetic construct (ie, at the 5' or 3 'end of the construct) in the same plasmid vector or can be co-transformed with the construct into a separate plasmid vector. Choices selectable markers are well known to those skilled in the art and include (synthetic or natural) genes conferring to the transformed cells the ability to utilize a metabolite that is not the amdS gene is normally metabolized by the microbe (e.g. of A. nidulans that codes for acetamidase and confers the ability to grow on acetamide as the sole source of nitrogen) or resistance to antibiotics (for example, the ph gene of Escheri chi a coli that codes for hygromycin-b-phosphotransferase and that confers hygromycin resistance). If the host strain lacks a functional gene for the chosen marker, then that gene can be used as a marker. Examples of those markers include trp ^ pyr4, pyrG, argB, l eu, and the like. The corresponding host strain will therefore have to lack a functional gene corresponding to the chosen marker, ie trp, pyr, a rg, l eu and the like. The selectable marker used in the genetic constructs described in Examples 5-7 is the E. coli hph gene expressed from the phosphoglycerate kinase (pgk) promoter of Tri choderma. The DNA sequence of the hph gene of E. coli can be found in Figure 4 of Gritz and Davies, "Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Es cheri chia col i and Saccharomyces cerevi siae ", Gene 25: 179-188, 1983; the DNA sequence of the pgk promoter of Tri ch oderma reesei can be found in Figure 2 of Vanhanen, Saloheimo, limen, Knowles and Penttila, "Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene (p? rkl) of Tri choderma reesei ", Gene 106: 129-133, 1991. A preferred embodiment of the invention comprises the genetic construction of beta-glucosidase so far described in this way. The practice of the present invention is not limited by the method of constructing the construct, which may include, but is not restricted to, normal molecular biology techniques such as isolation of plasmid DNA from E. coli by alkaline lysis, digestion of plasmid DNA with restriction endonucleases, separation and isolation of DNA fragments by agarose gel electrophoresis, ligation of DNA fragments with T4-DNA ligase, insertion of unique restriction sites at the ends of the DNA fragments by polymerase chain reaction or the addition of oligonucleotide linkers, and setting blunting of DNA fragments with T4-DNA polymerase or Klenow fragment of DNA polymerase I of E. coli Examples 1-7 describe methods for making these genetic constructs. In another preferred embodiment of the present invention, the genetic construction of beta-glucosidase is introduced and expressed in a microbial host to create a genetically modified microbe. The resulting genetically modified microbe produces an increased level of beta-glucosidase relative to the untransformed microbial host. The genetically modified microbe produces an increased level of beta-glucosidase of preferably about 10-fold relative to the untransformed microbial host, more preferably at least about 40-fold relative to the untransformed microbial host, and more preferably to the less about 120 times in relation to the untransformed microbial host. This invention encompasses any method for introducing the genetic construction of beta-glucosidase into the familiar microbial host by those skilled in the art, including, but not limited to, treatment with calcium chloride from bacterial cells or fungal protoplasts to weaken cell membranes, adding polyethylene glycol to allow fusion of cell membranes, which depolarization of cell membranes by electroporation, or firing of DNA through the cell wall and membranes via a bombardment of micro-projectiles with a particle gun. The example describes the procedures for introducing the genetic construction of beta-glucosidase into Tri ch oderma spores using a particle gun. The 10-fold improvement of beta-glucosidase production relative to the untransformed microbial host reflects a significant improvement that is well above the natural variability of the strain and is commercially significant. The degree of improvement of beta-glucosidase by this method has been as high as 126 times and could reach more than 1000 times. The measurement of the degree of improvement of beta-glucosidase production is by growing the culture and measuring the activity of the beta-glucosidase, as described in Example 11. It is believed that the genetic constructs of the present invention will produce any level of greater improvement of approximately 10 ve ce s. It is understood by those skilled in the art that the specific activity of beta-glucosidase of a mixture of enzymes (in IU / mg protein) is. it can increase by decreasing the amount of cellulase and other proteins in the enzyme mixture. This can be done by physical and mechanical separations of the enzyme mixture or by deletion of the cellulase or other genes by recombinant means. These methods have little or no effect on the actual production of beta-glucosidase by the microorganism. However, these methods may be optionally included in the practice of the present invention. In a preferred embodiment, the microbial host is a member of the species of Trichoderma, Humicola, Fusarium, Aspergillus, Streptomyces, Thermomonospora, Bacillus or Cellulomonas. These species are well suited because they produce cellulase in addition to beta-glucosidase. In addition, methods for the introduction of DNA construction into Trichoderma cellulase-producing strains have been published (Lorito, Hayes, DiPietro and Harman, 1993, "Biolistic Transformation of Trichoderma harzianum and Gliocladium virens using plasmid and geno ic DNA ", Curr. Genet 24: 349-356, Goldman, VanMontagu and Herrera-Estrella, 1990," Transformation of Trichoderma harzianum by high-voltage electric pulse ", Curr. Genet. 169-174; Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, "A versatile transformation system for the cellulolytic fungus Trichoderma reesei", Gene 6: 155-164), Aspergillus (Yelton, Hamer and Timberlake, 1984, "Transformation of Aspergillus nidulan s using a tprC plasmid ", Proc. Nati, Acad. Sci. USA 81: 1470-1474), Fusarium (Bajar, Podila and Kolattukudy, 1991," Identification of a fungal cutinase promoter that is inducible by a plant signal via a phosphorylated trans-acting factor ", Proc. Nati.

Acad. Sci. USA 88: 8202-8212), Streptomyces (Hopwood et al., 1985, "Genetic Manipulation of Streptomyces: a laboratory manual", The John Innes Foundation, Norwich, UK) and Bacillus (Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, "Genetic transformation of intact cells of Ba ci ll us subtili s' by electroporation", FEMS Microbio !. Lett 55: 135-138). The genetic constructs used in these published transformation methods are similar to those described in Examples 5-7 in that they contain a promoter linked to a protein coding region (which can encode a selectable marker) and a transcriptional terminator. In most cases, the genetic constructs are linked to a selectable marker gene. Although there are no published methods for the transformation of Humi col a, Th ermomon ospora or Cel ul omona s, it is believed that transformation methods for other fungi or filamentous bacteria can be used for strains Humi col a, Th erm omon ospora or Cell ul omona s by virtue of the similar morphologies and physiologies of these spices to those for which transformation methods have been published. In addition, transformation methods such as electroporation and particle bombardment have been used to introduce DNA into many different types of cells including mammalian and plant cells, bacterial, yeast and fungal cells. In a preferred embodiment, the xylanase secretion signal is native to the microbial host from which the genetically modified material is derived (i.e., the source of xylanase secretion signal is the same type of microbial host as the microbial host from the which genetically modified material is derived) .- In a more preferred embodiment, the microbial host is Tri choderma. In a more preferred embodiment, the microbial host is Tri ch oderma reesei.

EXAMPLES Example 1 describes the isolation of genomic DNA from the strains RutC30, M2C38 BTR48 of Tri choderma reesei and the genetically modified derivatives of these strains. Examples 2-7 describe the construction of genomic DNA libraries, the cloning of several genes, and various genetic constructs from strain M2C38, from Tri choderma rees ei. Examples 9 and 11-15 describe the transformation and expression of beta-glucosidase genetic constructs in strains M2C38 BTR48, and RutC30 from Tri ch oderma reesei. The strains M2C38 and BTR48 of Tri choderma reesei are patented strains of Iogen Corporation, and were derived from Tri choderma reesei RutC30 (ATCC 56765, Montenecourt and Eveleigh, 1979, "Selective isolation of high yielding cellulase mutants of T. Reesei", Adv. Chem. Ser. 181: 289-301), which in turn was derived from Tri choderma reesei Qm6A (ATCC 13631 Mandéis and Reese, 1957"Induction of cellulase in Tri choderma viride as influenced by coal sources and metais", J. Bacteriol. 73: 269-278). In Example 1 and the subsequent examples, restriction endonucleases, T4-DNA polymerase, T4-DNA ligase and the Klenow fragment of E. coli DNA polymerase 1 were purchased from Gibco / BRL, New England. Biolabs, Boehringer Mannheim or Pharmacia and were used as recommended by the manufacturer. The Pwo polymerase and the activity of reading-proof (Boehringer Mannheim) were used in all polymerase chain reactions (PCR) according to the manufacturer's protocol. Hygromycin B was purchased from CalBiochem.

EXAMPLE 1 Isolation of genomic DNA from Tri choderma reesei To isolate the genomic DNA, 50 ml of potato dextrose broth (Difco) was inoculated with T spores. reesei harvested from a potato dextrose agar plate with a sterile inoculation loop. The cultures were shaken at 200 rpm for 2-3 days at 28 ° C. The mycelia were filtered on a sterile GFA glass microfiber filter (Whatman) and washed with cold, deionized water. The fungal cakes were frozen in liquid nitrogen and ground in a powder with a pre-cooled mortar and pestle; 0.5 g of the powdered biomass was redispersed in 5 ml of 100 M Tris, 50 mM EDTA, pH 7.5 plus dodecyl-1% sodium sulfate (SDS). The lysate was centrifuged (5000 g for 20 minutes, 4 ° C) to pellet the cellular debris. The supernatant was extracted with a volume of buffer (10 mM Tris, 1 M EDTA, pH 8.0) saturated phenol followed by extraction with one volume of phenol: chloroform: isoamyl alcohol saturated with buffer (25: 24: 1) in order to remove the soluble proteins. The DNA was precipitated from the solution by adding 0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of 95% cold ethanol. After incubation for at least 1 hour at -20 ° C, the DNA was pelleted by centrifugation (5000 g for 20 minutes, 4 ° C), rinsed with 10 ml of 70% ethanol, air dried and redispersed in 1 mL of 10 mM Tris, 1 mM EDTA, pH 8.0. The RNA is digested by the addition of ribonuclease A (Boehringer Mannheim) added to a final concentration of 0.1 mg / ml and incubation at 37 ° C for 1 hour. Sequential extractions with a volume of phenol saturated with buffer and a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) saturated with buffer were used to remove the ribonuclease from the solution of DNA The DNA was precipitated again with 0.1 volumes of 3M sodium acetate, pH 5.2 and 2.5 volumes of 95% cold ethanol, sedimented by centrifugation, rinsed with 70% ethanol, air dried and redispersed in 50 μl of 10 mM Tris, 1 mM EDTA, pH 8.0. The concentration of DNA was determined by measuring the absorbance of the solution at 260 nm (p.Cl in Sambrook, Fritsch and Maniatis, "Molecular Cloning: A. Laboratory Manual, Second Edition", Cold Spring Harbor Press 1989, later referred to as Sambrook et al.).

EXAMPLE 2 Construction of Genomic Libraries of T. rees ei Two plasmid libraries and a phage library were constructed using the genomic DNA isolated from the M2C38 strain of T. rees ei. The plasmid libraries were constructed in the Pucll9 vector (Viera and Messing, "Isolation of single-stranded plasmid DNA", Methods Enzymol, 153: 3, 1987) as follows: 10 μg of genomic DNA was digested for 20 hours at 37 ° C in a volume of 100 μl with two units / μg of the restriction enzymes HindIII, BamHl or EcoRI. The digested DNA was fractionated on a 0.75% agarose gel run in 0.04 M Tris-acetate, 1 mM EDTA, and stained with ethidium bromide. Slices of gel corresponding to the sizes of the genes of interest (based on the published information of the Southern blots) were excised and subjected to electro-elution to recover the DNA fragments (Sambroo et al., Pp. 6.28-6.29 ).

These DNA enriched fractions were ligated into pUC119 in order to create gene libraries in ligation reactions containing 20-50 μg / ml of DNA in a 2: 1 mole ratio of vector: insert DNA, 1 mM ATP and 5 units of T4 DNA ligase in a total volume of 10-15 μl at 4 ° C for 16 hours. Strain HB101 from Es cheri chi a col i was electrophoresed with the ligation reactions using the Cell Porator System (Gibco / BRL) following the manufacturer's protocol and the transformants were selected on LB agar containing 70 μg / ml ampicillin. The phage library was constructed in the lambda vector, lambda DASH (Stratagene, Inc.) as follows: genomic DNA (3 μg) was digested with 2, 1, 0.5 and 0.2 units / μg of Bam Hl for 1 hour at 37 ° C to generate fragments of 9-23 kB in size. The DNA of each digestion was purified by extraction with a volume of phenol: chloroform: isoamyl alcohol saturated with Tris (25: 24: 1) followed by precipitation with 10 μl of 3M sodium acetate, pH 5.2 and 250 μl of 95% ethanol % (-20 ° C). The digested DNA was pelleted by microcentrifugation, rinsed with 0.5 ml of 70% cold ethanol, dried with air and redispersed in 10 μl of sterile, deionized water. Enrichment of fragments with a DNA size of 9-23 kB was confirmed by agarose gel electrophoresis (0.8% agarose in 0.04 M Tris-acetate, 1 mM EDTA). The digested DNA (0.4 μg) was ligated to 1 μg of lambda DASH arms predigested with BamHl (Stratagene) in a reaction containing 2 units of T4-DNA ligase and 1 mM ATP in a total volume of 5 μl at 4 ° C overnight. The ligation mixture was packed into phage particles using the GigaPack1 II Gold packaging extracts (Stratagene), following the manufacturer's protocol. The library was titrated using the host strain of E. coli XLl-Blue MRA (P2) and found to contain 3 x 10 5 independent clones.

EXAMPLE 3 Isolation of M2C38 clones from T. reesei of the cellobiohydrolase I (cbhl), cellobiohydrolase II (cbhl) and ß-glucosidase (bglI) genes from the libraries of pUC119 Transformants HB101 of E. col i having the clones cbh l, cbhl or bhl starting from the pUC119-Hind III, -BamHIo-EcoRI, recombinant libraries, were identified by the colony leaching hybridization: 1-3 x 104 colonies were transferred into membranes of HyBond ™ nylon (Amersham); the membranes were placed on the colony side up on transfer paper (VWR 238) saturated with 0.5 M NaOH, 1 M NaCl for 5 minutes to lyse the bacterial cells and denature the DNA. The membranes were then neutralized by placing the side of the colony upwards • on transfer paper (VWR 238) saturated with Tris 1.5 m, pH 7.5 plus 1 M NaCl for 5 minutes; the membranes were allowed to air dry for 30 minutes and the DNA was then fixed to the membranes by baking at 80 ° C for 2 hours. 32 P-labeled probes were prepared by PCR amplification of short fragments (0.7-1.5 kB) of the bgl l, cbh and cbhl coding regions from the enriched mixture of the Hind III, BamHl or EcoRI fragments, respectively, in a labeling reaction containing 10-50 ng of target DNA, d (GCT) TP, each 0.2 mM, dATP 0.5 μM, alpha-32P-dATP 20-40 μCi, 10 pmol of oligonucleotide primers and 0.5 units of Taq -polymerase in a total volume of 20 μl. the reaction was subjected to 6-7 cycles of amplification (95 ° C, 2 minutes, 56 ° C, 1.5 minutes, 70 ° C, 5 minutes). The amplified 32 P-labeled DNA was precipitated by the addition of 0.5 ml of trichloroacetic acid at 10% (w / v) and 0.5 mg of yeast tRNA. The DNA was pelleted by microcentrifugation, washed twice with 1 ml of 70% ethanol, dried with air and redispersed in 1 M Tris pH 7.5, 1 mM EDTA. The nylon membranes on which the pUC119 recombinant plasmids were fixed were prehybridized in thermally sealed bags for 1 hour at 60-65 ° C in 21 M NaCl, 1% SAS, 50 mM Tris, 1 mM EDTA, pH 7.5 with 100 μg / ml denatured, denatured salmon sperm DNA. Hybridizations were carried out in heat-sealed bags in the same buffer with only 50 μg / ml of salmon sperm DNA, stripped, denatured and 5 x 106-5 x 107 cpm of the bgl 1 probe, cbh and cbhl denatured for 16- 20 hours at 60-65 ° C. Membranes were washed once for 15 minutes with 1 M NaCl, 0.5% SDS at 60 ° C, twice for 15 minutes each with 0.3 M NaCl, 0.5% SDS at 60 ° C and once for 15 minutes with 0.03 M NaCl, 0.5% SDS at 55 ° C. The membranes were again placed in heat-sealed bags and exposed to a Kodak RP X-ray film at 16-48 hours at -70 ° C. The X-ray film was revealed following the manufacturer's protocols. Colonies that give strong or weak signals were harvested and cultured in 2xYT medium supplemented with 70 μg / ml ampicillin. Plasmid DNA was isolated from these cultures using the alkaline lysis method (Sambrook et al., Pp. 1.25-1.28) and analyzed by restriction digestion, Southern hybridization (Sambrook et al., Pp. 9.38- 9.44) and PCR analysis (Sambrook et al., Pp. 14.18-14.19). Clones having the bgl I gene were identified by the colony survey hybridization of the pUC119-Hind III library (Example 2) with a 1.0 kb bgll probe prepared using oligonucleotide primers designed to amplify 462-1403 bp of the sequence bgll published (Barnett et al.). A bgll clone, pJEN200, was isolated, containing a 6.0 kb Hind III fragment corresponding to the promoter, structural gene and termination sequences. Clones having the cbh1 gene were identified by colony-collation hybridization from the pUC119-Ba Hl library with 0.7 kb cbhl probes prepared using oligonucleotide primers designed to amplify 597-1361 bp of the published cbhl sequence (Shoemaker, Schweikart, Ladner, Gelfand, Kwok, Myambo and Innis, "Molecular cloning of exo-cellobiohydrolyase 1 derived from Tri choderma rees and strain L27", Bio / Technology 1: 691-696, 1983 hereafter referred to as Shoemaker et al. ). A cbhl clone, pCORl32, was isolated, which contains a BamHi fragment of 5.7 kb corresponding to the promoter (4.7 kb) and 1 kb of the structural gene cbhl. From this, a 2.5 kb EcoRI fragment containing the cbhl promoter (2.1 kb) and the 5 'end of the cbhl coding region (0.4 kb) was subcloned into pUC119 to generate pCB152. Clones that have the c <gene; h2 were identified by colony survey hybridization from the pUCH9-EcoRI library with a 1.5 kb cbhl probe prepared using oligonucleotide primers designed to amplify 580-2114 bp of the published cbhl sequence (Chen, Gritzali and Stafford, "Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Tri choderma reesei ", Bio / Technology 5: 274-278, 1987, hereafter referred to as Chen et al.). A cbhl clone, pZUKßOO, was isolated, containing an EcoRi fragment of 4.8 kb corresponding to the promoter (600 bp), structural gene (2.3 kb) and the terminator (1.9 kbp).

EXAMPLE 4 Cloning of the cbhl terminator of M2C38 of T. reesei, xylanase II gel (xlnl) phosphoglycerate kinase (pgk p) promoter. Probes labeled with digoxigen-11-dUTP were prepared from the PCR-amplified coding regions of the genes by random priming labeling using the DIG labeling and detection equipment (Boehringer Mannheim) and following the manufacturer's protocols. Genomic clones • containing the cbh l, xlnl and pgk genes were identified by plate-raising hybridization of the lambda library DASH For each gene of interest, 1 x 104 colonies were transferred to nylo Nytran® membranes (Schleicher and Schull). The phage particles were lysed and the phage DNA was denatured by placing the membranes on the side of the plate up in transfer paper (VWR238) saturated with 0.5 M NaOH, 1 M NaCl, for 5 minutes; the membranes were then neutralized by placing the membranes with the side of the plate up on the transfer paper (VWR238) saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 minutes; the membranes were allowed to air dry for 30 minutes and the DNA was then fixed to the membranes by baking at 80 ° C for 2 hours. The membranes were prehybridized in heat-sealed bags in a solution of 6X SSPE, 5X Denhardt, 1% SDS plus 100 μg / ml salmon sperm DNA, stripped, denatured at 65 ° C for 2 hours. The membranes were then hybridized in heat-sealed pouches in the same solution containing 50 μg / ml of stripped, denatured salmon sperm DNA and 0.5 μg of digoxigen-dUTP labeled probes at 65 ° C overnight. The membranes were washed twice for 15 minutes in 2X SSPE, 0.1% SDS at room temperature, twice for 15 minutes in 0.2 X SSPE, 0.1% SDS at 65 ° C and once for 5 minutes in 2X SSPE. The clones were positively hybridized and identified by reaction with an anti-digoxigenin / alkaline phosphatase antibody conjugate, 5-bromo-4-chloro-3-indoyl-phosphate and 4-nitro-blue-tetrazolium chloride (Boehringer Mannheim) following the manufacturer's protocol. The clones that hybridized positively were further purified by a second round of detection with the probes labeled with digoxigen-dUTP. Individual clones were isolated and the phage DNA was purified as described in Sambrook, et al., (1989) p. 2.118-2.121 with the exception that the CsCl gradient step was recoloured by extraction with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and one volume of chloroform: isoamyl alcohol (24: 1). The DNA was precipitated with 0.1 volumes of 3M sodium acetate and pH 5.2 and 2.5 volumes of 95% cold ethanol. The precipitated phage DNA was washed with 0.5 ml of 70% cold ethanol, dried with air and redispersed in 50 μl of 10 mM Tris, 1 mM EDTA, pH 8.0. Restriction fragments containing the genes of interest were identified by restriction digestion of purified phage DNA and Southern blot hybridization (Sambrook et al., Pp. 938-9.44) using the same probes with digoxigen-dUTP tags used to detect the lambda DASH library. The membranes were hybridized and the fragments positively hybridized were visualized by the same methods used for plaque removal. Once the desired restriction fragments of each lambda-DASH clone were identified, restriction digestions were repeated, the fragments were resolved on a 0.8% agarose gel in TAE and the desired bands were excised. The DNA was ligated from gel slices using the Sephaglas BandPrep Kit (Pharmacia) following the manufacturer's protocol. Clones having the cbhl gene were identified by hybridization with colony survey of the DASH lamda library (Example 2) with a cbhl probe comprising 45-2220 bp of the published cbhl sequence (Shoemaker et al.). A 1.8 kb BamHi fragment that contains the 3 'end of the cbh l coding region (0.5 kg) and the cbh l terminator (1.3 kb) was isolated by restriction digestion of the purified phage DNA from the lambda DASH clone cbh l. This fragment was subcloned into the BamH1 site of the plasmid vector pUC119 of E. col i to generate the plasmid pCBlTa. Clones having the gene xl.r¡2 were identified by hybridization of the colony of the lambda library DASH (Example 2) with a probe xlnl comprising 100-783 bp of the published sequence xlnl (Saarelainen, Paloheimo, Faberstrom, Suomioen and Nevalainen, "Cloning, sequencing and enhanced expression of the Tri choderma reesei endoxylanase II (pl 9) gene xln2", Mol. Gen. Genet. 241: 497-503, 1993, referenced later as Saarelainen et al.). A 5.7 kb Kpnl fragment containing the promoter (2.3 kb), the coding region (0.8 kb) of the terminator (2.6 kb), of the xlnl gene was isolated by restriction digestion of the purified phage DNA from the xlnl plan of lambda DASH. This fragment was subcloned into the Kpnl site of pUC119 to generate the plasmid pXYN2K-2. Clones having the pgkl gene were identified by colony-uptake hybridization from the lambda DASH library (Example 2) with a pgkl probe comprising 4-1586 bp of the pgk sequence published (Vanhanen, Penttila, Lehtovaara and Knowies, "Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) .from the filamentous fungus Tri choderma reesei ", Curr. Genet 15: 181-186 (1989) .A fragment of EcoRI DE 5.0 kb containing the promoter (2.9 kb) , coding region (1.6 kb) and the terminator (0.5 kb) of the pgk gene was isolated by restriction digestion of purified phage DNA from a pgk clone of lambda DASH This fragment was subcloned into the EcoRI site of pUC119 for generate the plasmid pGK5.0.

EXAMPLE 5 Construction of the β-glucosidase pCBGl-TV overexpression vector This example describes the construction of a vector containing the secretion signal and the cellobiohydrolase I promoter from Tri choderma and the mature beta-glucosidase coding region. A DNA fragment containing the bgl coding region minus the beta-glucosidase secretion signal (bp 474-2679) amplified by PCR from the pJEN200 template using primers homologous to the published bgll sequence containing either a Sphl site 5 'to Val32 of the encoded beta-glucosidase or a 3' site of Kpnl to the bgll terminator codon using PwO-polymerase this amplified fragment was digested with Sphl and Kpnl and inserted into pCB219N digested with Sphl and Kpnl to generate pBgstrf. To make pCB219N, a cbhl fragment was amplified from the pZUKdOO template using a pb homologous primer 2226-2242 of the published 3 'untranslated region of the cbhl gene (Chen et al., 1987) containing a Kpnl site at the 5' end and the Puc direct primer (Catalog No. 1224, New England Biolabs), which is fixed in the 3 'direction of the EcoRI site at the 3' end of cbh2 at Pzuk600. This fragment was digested at the Kpnl and EcoRI managed sites and inserted into the corresponding sites of pUC119 to generate pCB219. An EcoRI-Natl adapter (Catalog No. 35310-010, Gibco / BRL) was inserted into the unique EcoRI site of pCB219 to generate pCB219N. A fragment containing the cbhl promoter and the secretion signal was amplified from pCB152 using a specific cbhl primer (pb249-284 of the published cbhl sequence, Shoemaker et al., 1983) containing a 3 'sphl site to Serl9 of encoded cbh l and direct primer pUC (Catalog Number 1224, New England Biolabs) which is set in the 5 'direction of the EcoRI site at the 5' end of cbhl in pCB152. The PCR product of the cbh l promoter + the secretion signal was digested with sphl and EcoRI and inserted into the corresponding sites in pBR322L (a derivative of pBR322 in which the region between the sphl and Sali sites was replaced with a Sphl -Not I-Exit) to generate pBR322LCS. To make the expression cartridge, the bgll coding region and the cbhl terminator were isolated from pBgstrf as a 4.1 kb fragment of Sphl / Notl and inserted into pBR322LCS digested with Sphl and Notl. In order to maintain the correct reading frame at the junction of the mature cbhl and beta-glucosidase secretion signal, the resulting plasmid, pCBGstrf, was linearized at the unique Sphl site and the Sphl site was blunt-ended with Tr-DNA -polymer The resulting plasmid, pCBGl, was then further modified by conversion of the single Notl site at the 3 'end of the cbhl terminator to a unique Xhol site by the addition of Xhol linkers (Catalog Number 1073, New England Biolabs). The final plasmid, pCBGl-Xho, is the plasmid of the expression cartridge. The hygromycin-fosfotrans ferase gene (hph) of E.coli used as a selectable marker for T. reesei was amplified with Pwo polymerase from plasmid Pvul005 (Van den Elzen, townsend, Lee and bedbrook, • "A chi aeric hygromycin resitance gene as a selectable marker in plant cells", Plant Mol. Biol. 5: 299-302, 1989). The primers were digested to introduce the Sphl and Kpnl sites at the 5 'and 3' ends of the hph coding region (bp 211-1236 of the published hph sequence, gritz and Davies, "Plasmid-encoded hygromycin b resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Es cheri chi a col i and Sa ccharomyces cerevisia e "Gene 25: 179-188, 1983), respectively. the PCR product was digested with Sphl and Kpnl and inserted into the corresponding sites in the polylinker region of pUC119. The resulting plasmid, pHPTIOO was used as the starting plasmid for the construction of the selection cartridge. Two new linker regions were introduced into this plasmid to facilitate cloning of the promoter and terminator fragments. A HindI I-Xbaí ^ -Xhol-Sphl linker was inserted between the HindII and Sphl sites as well as a Kpnl-Not I-Sacl linker that was inserted between the Kpnl and Sacl sites of the pUC119 polylinker that remains in pHPTIOO. This construction was designated as pHPT102. The primers used to amplify the pgk promoter (Vanhanen, Saloheimo, Limen, Knowles and Penttila, "Promoter structure and expression of the 3-phosphoglycerate kinase gene (pgkl) of T. reesei", Gene 106: 129-133, 1991) were digested to introduce the Xhol site and a Sphl site in positions -970 to +1 of the promoter, respectively. these sites were subsequently used to insert the promoter pgken the Xhol and Sphl sites of pHPT102 to generate pHPT115. A cbh l determinant fragment of 1.3 kb was amplified with Pwo polymerase from pCBIT using a primer that is fixed to the 3 'introduced region of cbhl (bp 1864-1899 of the published cbhl sequence) containing a Kpnl site in pbl877 -1882 and the reversed pUC primer (Catalog Number 18432-013, Gibco / BRL) which is fixed in the 3 'direction of the EcoRI site at the 3' end of the cbh l terminator in pCBIT. The PCR product of the cbhl terminator was digested with Kpnl and inserted into the unique Kpnl site of pHPT115 to generate plasmid pHPT136 from the selection cartridge. To make the transformation vector, the expression cartridge from pCBGl-Xho was isolated as an Xbal / Xhol fragment of 5.6 kb and inserted between the unique Xbal and XhoLT sites in the 5 'direction of the pHPT136 selection cartridge . The final transformation vector, pCGl-Tv, as presented in Figure 1, was introduced as a circular plasmid in M2C38 of T. reesei via bombardment of microprojectiles as described later in Example 9.

EXAMPLE 6 Construction of beta-glucosidase pXBGl-TV overexpression vector This example describes the construction of a vector containing the Tri choderma xylanase II promoter and the secretion signal, and the mature beta-glucosidase coding region. The beta-glucosidase coding region (bp 474-2680) was amplified with Pwo polymerase from the genomic clone bgll, pJEN200 using primer to insert a Xbal site directly in the 5 'direction of pv 474 in the published bgll sequence ( Barnett, et al.) And a Kpnl site directly in the 3 rd direction of bp 2680. The blunt-ended PCR product was inserted into the Smal depUClld site to generate the designated plasmid, pBGm.s. Since the site Xbal was managed to be immediately in the 5 'direction of the onset of the mature beta-glucosidase in Bal32, the cloned fragment did not include the signal of beta-glucosidase secretion. Plasmid pBGm.s was digested with xbal and Kpnl and the 2.2 kb fragment containing the bgll coding region minus the secretion signal was isolated and inserted into the xbal and kpnl sites in the 5 'direction of the cbhl terminator in the plasmid pCB219N (described in Example 5, below), to produce the plasmid pBG2X. A 2.3 kb fragment containing the promoter and the secretion signal of the xlnl gene (pb -2150 to +99 where +1 indicates the ATG start codon) was amplified with Pwo polymerase from the genomic subclone xlnl pXYN2K-2 using a xlnl-specific primer containing a Nhel site directly in the 3 'direction of pb 103 of the published xlnl sequence (Saarelainen et al.) and the inverted pUC primer (Catalog Number 18432-013, Gibco / BRL) which is set to the 5 'direction of the Kpnl site at the 5' end of the xlnl gene. This xlnl PCR product was digested with EcoRI (which was amplified as part of the pUCl! 9 polylinker from pXYN2K-2) and Nhel and inserted into the plasmid pBR322L (described in Example 5 below) to generate pBR322LXN. The EcoRI site of pBR322LXN was then blunt-ended with Klenow and the Spel binders (Catalog Number 1086, New England Biolabs) were added to generate pBR322SpXN. Plasmid pBG2X was cut with Xbal and Notl and a 4.2 kb fragment, containing the gil coding region followed by the cbhl ending, was isolated. This fragment was inserted into the plasmid pBR322SpXN cut with Nhel and Notl '(Nhel and Xbal have compatible overhangs). This cloning resulted in fusion of the secretion signals of the xylanase directly to the mature beta-glucosidase creating the complete expression cartridge pXBG-2. The cbh l terminator in plasmid pHPT136 of the selection cartridge described in Example 5, above, was replaced with a 2.6 kb Kpnl fragment containing the transcriptional terminator xln2. The xln2 terminator was amplified with Pwo polymerase from the genomic subclone pXYN2K-2 using a primer to introduce the Kpnl site directly in the 3 'direction of pb 780 of the published xlnl sequence (Saarelainen et al.) And the forward primer pUC ( Catalog number 18431-015, Gibco / BRL) which is set in the 3 'direction of the 3' end of the xln2 gene in pXYN2K-2. The PCR product of the xlnl terminator was digested with Kpnl and ligated to a 5.1 kb Kpnl fragment from pHPT136 containing the hph gene promoted by pgk in pUC119 to generate plasmid pHPT136X from the selection cartridge. The construction of the transformation vector comprised the insertion of the expression cartridge directly in the 5 'direction of the pgk promoter from the selection cartridge. Plasmid pXBG2 of the expression cartridge was digested with Notl, the ends blunted using Klenow, and then digested with Spel. The pHPT136X expression cartridge was prepared in a similar manner by digesting with Xhol, followed by the filling in the reaction to create the blunt ends and then a digestion with Xbal. A blunt-adhesive ligation these two fragments were performed to ligate the blunt-ended Notl / Spel 6.5-kb fragment of pXBG2 to the blunt-ended Xbal / Xhol fragment of pHPT136 (Spel and Xbal have compatible overhangs). The final transformation vector, pXBG-TV, as depicted in Figure 2, was linearized in its unique N.otl before the M2C38 transformation of Tri choderma reesei via bombardment of microprojectiles, as described later in Example 9.

EXAMPLE 7 Construction of the pC / XBG overexpression vector (Xbal) -TV of beta-glucosidase This example describes the construction of a vector containing the cellobiohydrolase 1 promoter from Tri choderma, the xylanase secretion signal Ii and the coding region of mature beta-glucosidase. This example was carried out to test the combined effects of the cbhl promoter and the xlnl secretion signal in the bgl expression. A 1.2 kb HindIII fragment comprising pb -1399 to -204 of the cbhl promoter was amplified by PCR using the plasmid pBR322LCS (Example 5) containing the cbh1 promoter as a template in order to • to insert a unique Xbal site in bp -1393 a 1388. This modified cbhl promoter fragment was digested with HindIII and used to replace pb-1400 to -121 of the xl2 promoter in pxbgl (Example 6) to generate the new pC / XBG1 plasmid of expression cartridge. The 6.4 kb expression cartridge from pC / XBGl was isolated by digestion with No tl followed by transformation of blunt ends of the No tl site with the Klenow fragment and subsequent digestion with Spel. This fragment was then inserted by blunt-ended ligation / adhesiveness in the 5 'direction of the hph selection cartridge in pHPT136X which was digested with XhoI, blunt-ended in the Xhol site with Klenow and digested with Xbal. The final transformation vector, pC / XBG (CXbal) -TV (Accession No. 209613 deposit date February 3, 1988, American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA), as shown in Figure 3 was linearized with the unique Xbal and Notl sites at the 5 'end of the cbhl promoter and the 3' end of the xln2 terminator before the M2C38 transformation of T. reesei via bombardment of micropoiectiles, as described later in Example 9.

EXAMPLE 8 Southern blots of genomic DNA isolated from strains RutC30 and M2C38 of T. reesei Genomic DNA was isolated from each strain as described in Example 1. For Southern blotting, 1 μg of DNA was digested with 3-10 units of restriction endonuclease at 37 ° C for at least 2 hours and the digestion products were resolved on a 0.8% agarose gel in 0.04 M Tris-acetate, 1 mM EDTA. DNA was transferred by nylon membranes (Boehringer Mannheim) by capillary transfer (Sambrook et al., Pages 9.38-9.44). In Figures 4 and 5, lanes 2, 4, 6, 8, 10 and 12 contain digested M2C38 DNA and lanes 3, 5, 7, 9, 11 and 13 contain digested RutC30 DNA. The restriction endonucleases were BamHl (lanes 2 and 3), EcoRI (lanes 4 and 5), Xbal (lanes 6 and 7), HindIII (lanes 8 and 9), SstI (lanes 10 and 11), and Kpnl (lanes 12). and 13). In both figures, lane 1 contains lambda-Hindl I I molecular size standards (Gibco / BRL, Catalog Number 15612-013) and lane 14 contains 1 ng unlabeled fragment used to make the probe. The Southern blots were hybridized with a randomly primed probe labeled with digoxigen-11-dUTP prepared using the DIG labeling and detection equipment (Boehringer Mannheim). The template for the probe used in Figure 4 was a 2.3 kb fragment comprising the xlnl promoter of T. rees ei of the secretion signal (Saarelainen et al.). The template for the probe used in Figure 5 was a 2.1 kb fragment comprising pb 574-2679 of the mature coding region bgll of T. reeseí (Barnett, et al.). After the post-hybridization washes, the dig-dUTP complexes were visualized by incubation with an anti-digoxigenin alkaline phosphatase conjugate (Boehringer Mannheim) followed by reaction with 5-bromo-4-chloro-3-indoyl phosphate and 4-nitro-blue-tetrazolium chloride (Boehringer Mannheim).

EXAMPLE 9 Transformation of RutC30, M2C38 and BTR48 of T. microprojectile reuse The Biolistic PDS-1000 / He system (BioRad, E.I. DuPont de Nemours and Company) was used to transform spores of the RutC30, M2C38 and BTR48 strains of T. rees ei and all procedures were performed "as recommended by the manufacturer. M-10 tungsten particles (mean diameter of 0.7 μm) were used as microporters. The following parameters were used in the optimization of the transformation: a bursting pressure of 1100 psi, a helium pressure of 29 mm Hg, a separation distance of 0.95 cm, a macrocarrier travel distance of 16 mm, and a target distance of 9 c. Plates were prepared with 1 x 10d spores in potato dextrose agar (PDA) medium. The bombarded plates were incubated at 28 ° C. Four hours after the bombardment, the spores were subjected to primary selection by the selective PDA medium cover supplemented with 80 units / ml of HygB. The bombardment plates were incubated at 28 ° C. Transformants can be used after 3-6 days of growth. However, if additional incubation is necessary to achieve spoliation. After the spilling has occurred, a secondary selection process is performed to isolate the individual transformants. The spores are harvested from the plate with an inotion loop and redispersed in sterile water. This suspension is then filtered through a sterile syringe capped with glass microfibers. This allows the passage of spores while retaining unwanted mycelia. A determination of the concentration of spores in this suspension is required and subsequent dilutions are placed on plate on PDA plates supplemented with 0.75% Oxgall (Difco) and HygB (40 units / mL) to obtain 20-50 spores per plate. The Oxgall acts as a colony restrictor, thus allowing the isolation of individual colonies in these secondary selection plates. Isolated colonies can be observed after 2-3 days.

EXAMPLE 10 Southern blot analysis of genomic DNA isolated from strains RutC30, RC300, RC-302, M2C38, RM4-300, R4-301, RM4-302, BTR48, and RB4-301 of T. rees ei The genomic DNA of each strain was isolated as described in Example 1. For transfer Southern, 1 μg of DNA was digested with 3-10 units of Kpnl or Kbal at 37 ° C for at least 2 hours and the digestion products were resolved on a 0.8% agarose gel in 0.04 M Tris-acetate, EDTA 1 m. The DNA was transferred through nylon membranes (Boehringer Mannheim) by capillary transfer (Sambrook et al., Pp. 9.38-9.44). Southern blots were hybridized with a digoxigen-11-dUTP-labeled probe prepared using the DIG labeling and detection equipment (Boehringer Mannheim). The template was an EcoRI-Bgl II fragment of 1.3 kb comprising bp 1215-2464 of the published bgll sequence (Barnett et al.) After the post-hybridization washes, the dig-dUTP complexes were visualized by incubation with a conjugate of anti-digoxigenin alkaline phosphatase (Boehringer Mannheim) followed by reaction with the chemiluminescent reagent CSPD (Boehringer Mannheim) and exposure to X-ray film (Kodak). The results are summarized in Table 1.

TABLE 1 copy number of bgl in parenteral and recombinant T. reesei strains EXAMPLE 11 Production of β-glucosidase in liquid cultures This example describes the methods used to determine the amount of beta-glucosidase enzyme produced by a strain of Tri choderma. Individual colonies of Tri ch oderma were transferred to PDA plates for the propagation of each culture. Sporulation is required for uniform inoculation of the shake flasks that are used in the culture capacity test to produce beta-glucoside and cellulose. The culture medium is composed of the following: * Trace element solution contains 5 g / 1 of FeS04-7H20;, 1.6 g / 1 of MnS04-H20; 1.4 g / 1 of ZnS0 -7H20. ** 5 g / 1 more than 10 g / 1 benzoic globulate (when CBH or other cellulose promoter is used), 10 g / 1 silane (when the xlnl promoter is used), another carbon source compatible with the promoter that directs the expression of beta-glucosidase. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining medium. The liquid volume per 1 liter flask is 150 ml, the initial pH is 5.5 and each flask is sterilized by steam autoclave for 30 minutes at 121 ° C before inoculation. For both untransformed (i.e., native and transformed cells, spores are isolated from the PDA plates as described in Example 9 and 1-2 x 106 spores are used to inoculate each flask.) The flasks are shaken at 2100 rpm at a temperature of 28 ° C for a period of 6 days The filter containing the secreted protein was collected by filtration through microfiber glass filters GF / A (Whatman) The protein concentration was determined using the Bio-Rad protein assay (Catalog Number 500-0001) using Tri choderma cellulase as a standard. The beta-glucosidase activity is determined as described in Example 16. The transformants were selected for the ability to produce at least 10 times more beta-glucosidase (in IU / l) than the untransformed host strain as determined by the IU / l of the beta-glucosidase activity of the culture filtrate divided by the protein concentration (in mg / ml) the culture filtrate.

EXAMPLE 12 Production of beta-glucosidase by strains RurtC30, RC-300 and RC-302 using the Solka flocculation charcoal source Based on the previous facts using the cbhl promoter and the secretion signal to overexpress proteins in Tri ch oderma, the The mature beta-glucosidase coding region was placed in the 3 'direction of the cbh promoter and the secretion signal in the genetic construct shown in Figure 1 and described in Example 5 (pCBGl-TV). The vector was introduced into RutC30 of T. reesei by bombardment of particles (Example 9) and the resulting transformant RC-300, produced 7 times more beta-glucosidase activity than the strain of origin (Table 2). The 7-fold increase resulted from the incorporation of a copy of the transformation vector into the host chromosomes (Example 10, Table 1). The large increase in beta-glucosidase activity obtained from a copy of a construct in which beta-glucosidase is expressed using the cbh promoter and the secretion signal suggests that this strategy is better than that employed by Barnett et al. , and Fowler et al. which resulted in only a 5-fold increase in beta-glucosidase activity from 10-15 copies of a construct in which beta-glucosidase is expressed from its own promoter and secretion signal. However, the resulting 7-fold increase in beta-glucosidase activity was not yet sufficient to mitigate the beta-glucosidase deficit for cellulose hydrolysis. The RutC30 strain of T. Unreacted reesei was transformed with particle bombardment [Example 9]. by a genetic construct from the vector pC / XBG (Xbal) -TV that codes for the enzyme of beta-glucosidase of T. reesei matured enl hoe to the secretion signal xilanasa II of T. reesei. The untransformed RutC30 strain and the resulting transformed strain of this host, RC-302, were cultured using the procedures of Example 1 with 10 g / L of Solka flocculation and 5 μL of glucose as carbon sources. The results are shown in Table 2. The untransformed strain produced 0.14 IU of beta-glucosidase per mg of protein. The transformant RC-302 with the cbhl promoter and the xylanase II secretion signal produced 19 IU / mg of beta-glucosidase. This represents close to a 136-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process. The transformant RC-302 with the cbhl promoter and the xylanase II secretion signal produced about 19 times more beta-glucosidase activity than the best transformant of RutC30 with the cbhl promoter and the cbhl secretion signal.

TABLE 2 Production of ß-glucosidase in strains RutC30, RC-300 and RC-302 of T. reesei in 150 ml flask cultures EXAMPLE 13 Production of beta-glucosidase by strains M2C38 and RM4-302 using the carbon source of flocculation of Solka The vector pCBGl-TV, in which the beta-glucosidase is expressed from the promoter of the cbhl promoter and the secretion signal (Figure 1 and Example 5) was introduced into T. reesei M2C38 by particle bombardment (Example 9). The resulting transformant RM4-300 produced about 7-12 times more beta-glucosidase activity than the parent strain (Table 3). The untransformed T. reesei strain M2C38 is transformed by particle bombardment (Example 9) with a genetic construct of the vector pC / XBG (Xbal) -TV coding for the beta-glucosidase enzyme of T. reesei, mature linked to the secretion signal of xylanase II of T. reesei The untransformed strain M2C38 and the transformed strain of this host, RM4-302, were cultured using the procedures of Example 11 with 10 g / L of Solka flocculation and 5 g / L of glucose as carbon sources. The results are shown in Table 3. The untransformed strain produced 0.35 IU of b per mg of protein. The transformant RM4-302 with the cbh1 promoter and the xylanase II secretion signal produced 14.1 IU / mg beta-glucosidase. This represents about a 40-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process. The transformant RM4-302 with the cbhl promoter and the xylanase II secretion signal produced about three times more beta-glucosidase activity than the transformant with the cbh1 promoter and the cbh1 secretion signal. This is a significant difference, since the cbhl promoter and the secretion signal do not lead to sufficient production of beta-glucosidase to completely suppress the production of cellobiose in hydrolysis.

TABLE 3 Production of ß-glucosidase in strains M2C38, RM4-300 and RM4-302 of T. reesei in 150 ml flask cultures EXAMPLE 14 Production of beta-glucosidase by strains M2C38 and RM4-301 of T. reesei using the xylan coal source The M2C38 strain of T. Unreacted reesei was transformed by particle bombardment (Example 9) with a genetic construct of the pXBGl-TV vector encoding the beta-glucosidase of T. mature reesei linked to the xylanase promoter and the secretion signal.

The untransformed signal M2C38 and a strain transformed from this host, RM4-301, were cultured using the procedures of Example 11 with 5 g / L glucose and 10 g / L xylan as the carbon source. The results are shown in Table 4. The untransformed strain produced 0.16 IU of beta-glucosidase per mg of protein. The transformant RM4-301 with the xylanase II promoter and the xylanase II secretion signal produced 20.4 IU / mg beta-glucosidase. This represents about a 127-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process.

TABLE 4 Production of ß-glucosidase in strains M2C38 and RM4- 301 of T. reesei with xylan in 150 ml flask cultures EXAMPLE 15 Production of beta-glucosidase by BTR-48 strains and RB48-301 using the Solka flocculation carbon source The BTR48 strain of T. rees ei untransformed was transformed by bombardment of particles with a genetic construct from the vector pXBGl-TV that codes for the beta-glucosidase of T. mature reesei linked to the xylanase promoter and the secretion signal. The untransformed strain BTR-48 and a strain transformed from this host, RB48-301, were cultured using the procedures of Example 11 with 5 g / L and 10 g / L flocculation of Sslka as the carbon sources. The results are shown in Table 5. The untransformed strain produced 0.16 IU of beta-glucosidase per mg of protein. The transformant RB48-301 with the xylanase II promoter and the xylanase II secretion signal produced about 21.9 IU / mg beta-glucosidase. This represents close to a 136-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process.

TABLE 5 Production of beta-glucosidase in strains BTR48 and RB48-301 of T. reesei with Solka flocculation in 150 ml flask culture EXAMPLE 16 Measurement of beta-glucosidase activity of an enzyme mixture The beta-glucosidase activity of a semi-synthetic enzyme using the Ghose methods, "Measurement of Cellulase Activities", Puré and Appl. Chem., 59: 257-268 (1987), as follows. The enzyme sample is diluted to various concentrations in 50 mM sodium citrate buffers, pH 4.8, at a volume of 0.5 ml. A convenient range of dilutions is 3 to 24 times the estimated activity of the sample. For example, a sample of 10 units / ml should be diluted 1:30 to 1:40. In spite of the dilutions used, a 0.5 ml sample of citrate buffer is added to each enzyme tube. The substrate is prepared as a 15 mM cellobiose (5.3 g / L). The diluted enzyme is stored and the substrate is preheated separately at 50 ° C for 5 minutes, then an aliquot of 0.5 ml of the substrate is added to each tube with the enzyme. The tubes are tested and incubated for 30 minutes at 50 ° C. The reaction is terminated by immersing each tube in a boiling water bath for 5 minutes. The tubes are then mixed with a vortex, and the amount of sugar produced by each enzyme sample is measured on a YSI glucose analyzer, taking into account the small background of the enzyme. • One unit of beta-glucosidase activity is defined as the number of micromoles of glucose produced per minute. The activity is calculated based on equation 1 using the average value of each of the dilutions that produce 0.15 to 1.5 mg / ml of glucose.

A = C * G * D (1) where A = activity, beta-glucosidase units / ml (or micromoles of glucose / ml / min) C = 16.7 micromoles / mg / min G = glucose produced, mg / ml D = enzyme dilution, without dimensions EXAMPLE 17 Cellulose Hydrolysis The purpose of this experiment was to demonstrate the effectiveness of the beta-glucosidase elaborated by the transformed Tri-choderma by improving cellulose hydrolysis. The enzymes used for this study were Celulasa logen, a commercial cellulase enzyme from logen Corporation, and the RM4-302 product grown in a 30 liter fermentation vessel using the procedures described in example 11, with twice the levels of media concentration on that example. The enzymatic concentration was increased by ultrafiltration through an Amicon 10,000 MWCO membrane and normalized to the same cellulase activity as the Celulase de logen. The activities of these two enzymes are shown in Table 6.

TABLE 6 Enzymatic activities used in the cellulose hydrolysis study The celluloses used for this study were pre-treated oat husks, prepared according to the procedures of Foody, et al, Improved Pretreatment Process for Conversion of Cellulose to Fuel Ethanol, United States patent application filed on June 9, 1997, Example 6. The pre-treated oat husk cellulose samples of 0.5 grams were added to 25 ml flasks with 49.5 grams of a 0.05 molar sodium citrate buffer, pH 4.8. Enzymes were added to the flask: in an amount corresponding to 10 FPU per gram of cellulose. The resulting doses of beta-glucosidase are listed in Table 6. In both cases, the flasks were shaken at 250 RPM and kept at 50 ° C for 24 hours. At that time, samples were taken, filtered to remove the insoluble cellulose, and analyzed for the concentration of glucose and cellobiose using normal HPLC amperometric HPLC carbohydrate analysis methods. The results are listed in Table 7. Cellulase logen, the conventional cellulase of Tri choderma, converted only 45% of cellulose to glucose. This is unacceptably low for an ethanol process. The accumulation of cellobiose was significant, representing 13% of the cellulose. 'Cellulase with improved beta-glucosidase performed much better. The conversion of cellulose to glucose reached 84%. The reason for this excellent performance was that the accumulation of cellobiose was negligible, due to the abundance of beta-glucosidase.

TABLE 7 Hydrolysis of cellulose improved by high beta-glucosidase EXAMPLE 8 Comparison of xln2 and bgl1 genes from Tri ch oderma reesei in strains and M2C38 Southern blot analyzes were performed on the DNA of M2C38 and RutC30 digested with six different restriction enzymes that cut both inside and outside the regions they code for. the mature beta-glucosidase and the xylanase secretion signal (Example 8) to determine if there is any polymorphism between the two strains. As shown in Figures 4 and 5, identical bands were found that hybridize with the labeled probes prepared for fragments of M2C38 that encode the mature beta-glucosidase enzyme and the xylanase II promoter plus the secretion signal, not indicating polymorphisms and a high degree of DNA sequence homology in these regions between the two strains. The waves and primers used to identify and clone the M2C38 DNA sequences necessary to make the genetic constructs described in Examples 5-7 were based on the published DNA sequences of the various genes from several different strains of Tri choderma reesei including QM9414 (pgk, Vanhanen et al., 1989 and cbhl, Chen et al.), derivatives VTT-D / 79125 of QM9414 (xlnl_, Saarelainen et al.) and L27 (cbhl, Shoemaker et al.), and strain P40 (bgl I, Barnett et al.) derived from strain RL-P37. All these strains, type M2C38, are derived from the strain QM6a (Carter, Allison, King and Dunn-Coleman, "Chromosomal and genetic analysis of the electrophoretic karyotype of Tri-chorema rees ei: mapping of the cellulase and xilanase genes," Molecular Microbiology 6: 2167-2174, 1992). Because RutC30 is the parent derivative of QM6a of M2C38, the inventors are confident that the method as described in Examples 2-4, for the isolation of the gene sequences used to make the beta-glucosidase expression vectors described in Examples 5-7 will work equally well for the isolation of the same gene sequences from both M2C38 and RutC30. Based on the lineage of the strain described above and the Southern blot data, the inventors also have a high degree of confidence that the gene products prepared from RutC30 DNA will contain the identical DNA segments encoding the beta-enzyme. mature glucosidase and the secretion signal of xylanase II as those prepared from the DNA of M2C38. since the constructs prepared from the M2C38 DNA (Examples 5-7) result in an improved expression of beta-glucosidase in both M2C38 and RutC30 (Examples 12-14), the inventors are also confident that the genetic constructs elaborated Starting from the RutC30 DNA will result in similar levels of improvement of beta-glucosidase activity in both RutC30 and M2C38. While the present invention has been described with respect to what is currently considered to be the preferred modalities, it is to be understood that the invention is not limited to the embodiments described. On the contrary, the invention is proposed to cover several modifications and equivalent arrangements included within the spirit and scope of the appended claims. The scope of the following claims is to grant the broadest interpretation to encompass all equivalent modifications and formulations and functions.

Claims (27)

1. CLAIMS 1. A genetic construct comprising: a promoter selected from the group consisting of the cbhl, cbhl, egl, egl, e 3, eg5, xlnl and xlnl promoters of Tri choderma, in operative association with a xylanase secretion signal of a xylanase gene of Family 11, and a mature beta-glucosidase coding region of a beta-glucosidase gene selected from the group consisting of Tri-choderma beta-glucosidase genes, Aspergillus,
2. Humicola and Fausarium. The genetic construct according to claim 1, wherein the xylanase gene of Family 11 comprises a Tri choderma xylanase gene.
3. 3. The genetic construct according to claim 2, wherein the xylanase gene of Tri ch oderma comprises a xylanase I gene of Tri choderma by a xylanase II gene of Tri choderma.
4. The genetic construct according to claim 3, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
5. 5. The genetic construct according to claim 4, wherein the Tri-choderma beta-glucosidase gene comprises a bgl1 gene from Tri choderma.
6. 6. A genetically modified microbe comprising: a microbe selected from the group consisting of Tri choderma, Humi cola, Fusari um, Streptomyces, Thermomonospora, Ba cil l us

   Cell ulmonas, and Aspergillus, and the genetic construct of claim 1 that has been introduced into the microbe, wherein the genetically modified microbe produces an increased level of beta-glucosidase relative to the microbe.
7. 7. The genetically modified microbe according to claim 6, wherein the microbe is a Tri choderma microbe.
8. 8. The genetically modified microbe according to claim 7, wherein the Trichoderma microbe is a microbe of Tri choderma reesei.
9. 9. The genetically modified microbe according to claim 6, wherein the genetically modified microbe produces an increased level of beta-glucosidase of at least about 10 times.
10. The genetically modified microbe according to claim 6, wherein the genetically modified microbe produces an increased level of beta-glucosidase of at least about 40 times.
11. The genetically modified microbe according to claim 6, wherein the genetically modified microbe produces an increased level of beta-glucosidase of at least about 120 fold.
12. The genetically modified microbe according to claim 6, wherein the xylanase secretion signal is native to the microbe from which the genetically modified microbe is derived.
13. The genetically modified microbe according to claim 6, wherein the xylanase secretion signal comprises a xylanase secretion signal from a family of xylanase gene 11.
14. The genetically modified microbe according to claim 13, wherein the The xylanase gene of Family 11 comprises a xylanase gene from Tri choderma.
15. 15. The genetically modified microbe according to claim 14, wherein the Tri choderma xylanase gene comprises a xylanase I gene from Tri ch oderma or a xylanase II gene from Tri choderma.
16. 16. The genetically modified microbe according to claim 14, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a bgll gene of Tri choderma, and wherein the promoter is selected from the group consisting of promoter cbhl, cbhl, egl, egl, eg3, eg5, xlnl and xln2 of Tri choderma.
17. 17. The genetically modified microbe according to claim 6, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
18. 18. The genetically modified microbe according to claim 1, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
19. 19. The genetically modified microbe according to claim 8, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
20. The genetically modified microbe according to claim 9, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
21. The genetically modified microbe according to claim 10, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma. eleven .
22. The genetically modified microbe according to claim 11, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
23. 23. The genetically modified microbe according to claim 12, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene from Tri choderma.
24. 24. The genetically modified microbe according to claim 13, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene, from Tri choderma.
25. 25. The genetically modified microbe according to claim 14, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene of Trichoderma.
26. 26. The genetically modified microbe according to claim 15, wherein the mature beta-glucosidase coding region comprises a mature beta-glucosidase coding region of a beta-glucosidase gene of Tri choderma.
27. 27. A method for producing beta-glucosidase, comprising: transforming a microbe with the genetic construct of claim 1 to create a genetically modified microbe; and using the genetically modified microbe to produce an increased level of beta-glucosidase relative to the microbe before it is transformed. The method according to claim 27, wherein the transformation step comprises transforming a microbe selected from the group consisting of Tri choderma Humi col a, Fusari um,

    - Streptomyces, Th ermomonospora, Ba cill us Cel l ul omona s, and Aspergi ll us, to create a genetically modified microbe. The method according to claim 27, wherein the step of use comprises using the genetically modified microbe to produce an increased level of beta-glucosidase of at least about 10-fold. 30. The method according to claim 27, wherein the step of use comprises using the genetically modified microbe to produce an increased level of beta-glucosidase of at least about 40-fold. The method according to claim 27, wherein the step of use comprises using the genetically modified microbe to produce an increased level of beta-glucosidase of at least about 120 fold.