Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Definitions

• This invention relates to exoglucanases. More specifically, it relates to Trichoderma reesei cellobiohydrolase I reduced glycosylation variants which enable expression of the active enzyme in a heterologous host.
• pretreatment heats the substrate past the phase-transition temperature of lignin; and (2) pretreated biomass contains less acetylated hemicellulose.
• pretreated biomass contains less acetylated hemicellulose.
• pretreated biomass contains displaced and modified lignin. This alteration results in a non-specific binding of the protein with the biomass, which impedes enzymatic activity.
• pretreated biomass is a hardwood-pulp it contains a weak net-negatively charged surface, which is not observed in native wood. Therefore, for the efficient production of ethanol from pretreated biomass it is desirable to enhance the catalytic activity of glycosyl hydrolases on acid hydrolyzed hardwoods.
• Trichoderma reesei CBH I is a mesophilic cellulase enzyme, and comprises a major catalyst in the overall hydrolysis of cellulose.
• An artificial ternary cellulase system consisting of a 90: 10:2 mixture of T. reesei CBH I, A. cellulotyticus ⁇ l, and A. niger ⁇ -D-glucosidase is capable of releasing as much reducing sugar from pretreated yellow poplar as the native T. reesei system after 120 h.
• T. reesei CBH I variant enzymes capable of active expression in a heterologous host.
• the heterologous host Aspergillus awamori could provide an excellent capacity for synthesis and secretion of T. reesei CBH I because of its ability to correctly fold and post-translationally modify proteins of eukaryotic origin.
• A. awamori is believed to be an excellent test-bed for Trichoderma coding sequences and resolves some of the problems associated with direct site directed mutagenesis in Trichoderma.
• Another object of the invention is to provide a variant exoglucanase characterized by a reduction in glycosylation when expressed in a heterologous host.
• Another object of the invention is to provide an active cellobiohydrolase enzyme capable of expression in heterologous fungi or yeast. It is yet another object of the invention to provide a method for reducing the glycosylation of a cellobiohydrolase enzyme for expression in a heterologous host.
• the invention provides a method for making an active exoglucanase in a heterologous host, the method comprising reducing glycosylation of the exoglucanase, reducing glycosylation further comprising replacing an N-glycosylation site amino acid residue with a non- glycosyl accepting amino acid residue.
• the invention further provides a cellobiohydrolase, comprising the reduced glycosylation variant cellobiose enzymes CBHTN45 A; CBHIN270A; or CBHIN384A, or any combination thereof.
• a method for reducing the glycosylation of an expressed Trichoderma reesei CBHI protein by site-directed mutagenesis is disclosed.
• the method includes replacing an N- glycosylation site amino acid residue, such as asparagines 45, 270, and/or 384 of SEQ. ID NO: 4 (referenced herein as CBHTN45A, CBHIN270A and CBHTN384A, respectively), with a non- glycosyl accepting amino acid residue, such as is alanine.
• Various mutagenesis kits for SDM are available to those skilled in the art and the methods for SDM are well known.
• CBHIN45A discloses a procedure for making and using CBHI variants: CBHIN45A; CBHIN270A; and CBHIN384A.
• the examples below demonstrate the expression of active CBH I in the heterologous fungus Aspergillus awamori.
• Trichoderma reesei Trichoderma reesei.
• the cbhl genes included both cDNA and genomic (intron containing) versions. These were altered by site-directed mutagenesis for the specific purpose of reducing the glycosylation of the expressed CBH I protein through replacement of the N-glycosylation site amino acid residues (asparagine) with non-glycosyl accepting amino acid residues (alanine).
• the gene was propagated in an E. coli vector plasmid (pPFE2) under the control of the Aspergillus awamori glucoamylase promoter and signal sequence, and trpC terminator, and carrying resistance to ampicillin (E. coli selection) and Zeocin (Bleomycin) Aspergillus selection.
• CBHIN270A SEQ. ID. NO: 2
• CBHIN270A SEQ. ID. NO: 2
• CBHIN45A SEQ. ID. NO: 1
• CBHTN384A SEQ. ID. NO: 3
• Example 1 Production of Active Recombinant CBH I (rCBH I) in Aspergillus awamori Construction of Modified CBH I Coding Sequence.
• the coding sequence for T. reesei CBH I (SEQ. ID. NO: 4) was successfully inserted and expressed in Aspergillus awamori using the fungal expression vector pPFE2 (and pPFEl).
• Vectors pPFEl and pPFE2 are£. coli-Aspergillus shuttle vectors, and contain elements required for maintenance in both hosts. They encode ampicillin resistance for selection in E. coli and Zeocin resistance for selection in Aspergillus.
• the foregoing provided for the site-directed mutagenesis in E. coli, followed by expression of the new mutant proteins in A. awamori.
• the foregoing provided for the site-directed mutagenesis in E. coli, followed by expression of the new mutant proteins in A. awamori.
• CBH I gene is under the control of the A. awamori glucoamylase promoter and includes the glucoamylase secretion signal peptide.
• the construction of this plasmid required the addition, by PCR, of a Notl site and Xbal site on the coding sequence of CBH 1.
• the Notl site addition resulted in a change of the most N- terminal amino acid on the protein from glutamine to glycine. This glycine was subsequently changed back to the native glutamine in the pPFE2/CBHI construct, using site-directed mutagenesis PCR. This new construct was used to transform A.
• rCBH I rCBH I expressed in A. awamori tends to be over glycosylated as evidenced by the higher molecular weight observed on western blot analysis.
• Over-glycosylation of CBH I by A. awamori was confirmed by digestion of the recombinant protein with endoglycosidases. Following endoglycosidase H and F digestion, the higher molecular weight form of the protein collapses to a molecular weight similar to native CBH 1.
• the vector pPFE2/CBHI requires a relatively long PCR reaction (8.2 kb) to make site- specific changes using the Stratagene Quik Change protocol.
• the PCR reaction was optimized as follows using a GeneAmp PCR System 2400, Perkin Elmer Corporation.
• the reaction mixture contained 50 ng of template DNA, 125 ng each of the sense and antisense mutagenic primers, 5 ⁇ l of Stratagene lOx cloned Pfu buffer, 200 ⁇ M of each: dNTP, 5 mM MgCl 2 (total final concentration Of MgCl 2 is 7 mM); and 2.5 U Pfu Turbo DNA polymerase.
• the PCR reaction was carried out for 30 cycles, each consisting of one minute denaturation at 96C°, 1 minute annealing at 69°C, and 20-minute extension at 75°C. There is an initial denaturation for 2 minutes at 96°C and a final extension for 10 minutes at 75°C, followed by a hold at 4°C. Agarose gel electrophoresis, ethidium bromide staining, and visualization under UV transillumination were used to confirm the presence of a PCR product.
• PCR products were digested with restriction enzyme Dpnl, to degrade un-mutagenized parental DNA, and transformed into E. coli (Stratagene Epicurian Coli Supercompetent XL-1 Cells). Amp R colonies were picked from LB-Amp 100 plates and mutations were confirmed by DNA sequencing. Depending on scale, plasmid DNA was purified using the Qiagen QiaPrep Spin Miniprep Kit or the Promega Wizard Plus MaxiPrep DNA Purification System.
• spores were inoculated into 50 mL CM maltose medium, pH 5 0, and grown at 32°C, 225 rpm in 250 mL baffled flasks The cultures were transferred to 1 0 L of CM maltose in 2,800 mL Fernbach flasks and grown under similar conditions For large-scale enzyme production (>1 mg), these cultures were transferred to 10-L CM maltose in a New Brunswick BioFlo3000 chemostat (10-L working volume) maintained at 20% DO; pH 4 5, 25°C, and 300 rpm. The culture was harvested by filtration through Miracloth after 2-3 days of growth.
• the filtrate was concentrated and dia-flltered into 20mM sodium acetate pH 5.0 by tangential flow ultrafiltration with an Amicon DC30 concentrator equipped with a single 10,000 MWCO hollow fiber cartridge (1.1mm I D., 2.4 m 2 surface area).
• the retentate from the 10-L concentration or the filtrate from smaller cultures was clarified in an
• the permeate was further concentrated with an Amicon CH-2 concentrator equipped with three 10,000 MWCO hollow fiber cartridges (1.1 mm I D., 0.03 m 2 surface area). The final concentrate was sterile filtered through a 0.45 ⁇ m filter and stored at 4°C until used.
• the recombinant CBH I protein SEQ. ID NO: 4, was purified by passing the concentrated culture broth over two or three CBinD900 cartridge columns (Novagen, Madison,
• the eluted rCBH I was concentrated in an Amicon 10 mL stirred cell using a 25 mm PM10 membrane to ⁇ 2.0 mL and loaded onto a Pharmacia SuperDex200 16/60 size-exclusion column.
• the mobile phase was: 20 mM sodium acetate; 100 mM sodium chloride; and 0.02% sodium azide, pH 5.0 running at 1.0 mL/min.
• the eluted protein was concentrated by stirred cell and stored at 4°C. Concentration was determined by A 28 o using the extinction coefficient and molecular weight calculated for individual proteins by the ProtParam tool on the ExPASy website
• Example 2 Production of Reduced Glycosylation rCBH 1: Sites N270A: N45A: and N384A.
• rCBHl/pPFE2 has been optimized using site-directed mutagenesis to achieve expression of native molecular weight CBH I in A awamori.
• the QuickChange SDM kit (Stratagene, San Diego, Ca) was used to make point mutations, switch amino acids, and delete or insert amino acids in the native CBH1 gene sequence.
• the Quick Change SDM technique was performed using thermotolerant Pfu DNA polymerase, which replicates both plasmid strands with high fidelity and without displacing the mutant oligonucleotide primers.
• the procedure used the polymerase chain reaction (PCR) to modify the cloned CBH1 DNA.
• the basic procedure used a supercoiled double stranded DNA (dsDNA) vector, with an insert of interest, and two synthetic oligonucleotide primers containing a desired mutation.
• the oligonucleotide primers each complimentary to opposite strands of the vector, extend during temperature cycling by means of the polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks was generated. Following temperature cycling, the product was treated with a Dpnl restriction enzyme.
• Dpnl is specific for methylated and hemi-methylated DNA and thus digests the unmutated parental DNA template, selecting for the mutation-containing, newly-synthesized DNA.
• Three glycosylation-site amino acids on the protein surface were targeted for substitution of an alanine (A) residue in place of asparagine (N). Single site substitutions were successfully completed in the CBH I coding sequence at sites N45, N270, and N384, of SEQ. ID NO.: 4 by site-directed mutagenesis, and confirmed by DNA sequencing.
• Table 1
• variants CBHIN45A SEQ. ED NO: 1
• CBHI384A SEQ. ED NO.: 3
• Double and triple combinations of this substitution have also been completed in the CBH I coding sequence (SEQ. ED NO.: 4) at sites N45, N270, and N384 by site-directed mutagenesis and confirmed by DNA sequencing. These double and triple-site constructs will also yield rCBH I enzymes with reduced glycosylation and, presumably, native activity.
• GGC AAATGTGATTTCC AAGCGCC AGTCGGCCTGC ACTCTCC antisense strand

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=22505256

Family Applications (1)

Country Status (4)

Families Citing this family (10)

Family Cites Families (5)

• 2000
  • 2000-07-13 EP EP00948637A patent/EP1200574A4/de not_active Withdrawn
  • 2000-07-13 CA CA002379326A patent/CA2379326A1/en not_active Abandoned
  • 2000-07-13 AU AU62108/00A patent/AU6210800A/en not_active Abandoned
  • 2000-07-13 WO PCT/US2000/019007 patent/WO2001004284A1/en not_active Application Discontinuation

Also Published As

Similar Documents

Legal Events