Industrial Agricultural Biotech – Andre J. Laroche, Timothy Y. Huang, Michele M. Frick, Zhen-Xiang Lu, Hung Chang Huang, Kuo Joan Cheng, Agriculture and Agri Food Canada AAFC
Abstract for “Coniothyrium minitans xylanase gene Cxy1”
“The nucleotide sequence for a novel xylanase enzyme, denoted Cxy1, was obtained from Coniothyrium miniaturetans soilborne fungus. It also contains the amino acid sequence for the encoded xylanase, Cxy1.
Background for “Coniothyrium minitans xylanase gene Cxy1”
Cell wall structure and rigidity are achieved by the presence of cellulose, hempicellulose, and other lignin polymers. Complex carbohydrates are essential to maintain physical integrity and prevent plant pathogens from infecting your cells. Xylan is a polymeric chain made up of xylose carbohydrate monomers linked with successive?-1.4 xylose linkages. It makes up a large portion of plant hemicellulose. Xylan is responsible for the strength of plant secondary walls and limits the access of cellulases to cells wall. This prevents plant surface damage from plant pathogens.
“The hydrolysis of xylan is a key step in the degradation of plant tissues. This enzyme is produced by fibrolytic bacteria or fungi. A consortium of hydrolytic enzymes is responsible for xylan’s complex physical structure. Endoxylanlolytic enzymes (EC 3.2.1.8) break down xylan polymers into shorter oligoxylan pieces. The resulting oligoxylan bits are then degraded by?xylosidase (EC 3.2.1.37) (Bieley 1985). The hydrolysis of xylan can be complicated by various substituents, such as acetyl and arabinosyl along the elongated,?-1.4 xylose chain. The presence of different substituent groups in xylan polymers can vary between plant types. Xylan from grasses appears to be dominated by arabinose constituents. However, xylan polymers from hardwoods have acetyl- and arabinose substitutes. Endoxylanolytic hydrolysis is impeded by the steric impediment caused by these substituents attached the the?-1.4 xylan backbone. Xylanolytic degradation is therefore effected by xylanases in concert with enzymes such as ?-L-arabinofuranosidases (EC 3.2.1.55) and acetylesterases (EC 3.1.1.6), which cleave substituents from the xylan polymer.”
There are many applications for xylanolytic enzymes in biotechnology and industry. These include the addition of xylanases to biopulping processes. The enzymatic exclusion of xylan tissue from woody plants may improve pulp quality. Cellulases and/or xylanases can be used to improve the nutritional availability of feed carbohydrate and fermentative composting by predigesting forage crops. Xylanases can also be used to reduce organic waste disposal at landfill sites. Xylanases can also be used to improve quality of flour for baked goods. Xylan is a significant reserve of nutritive sugar in plant biomass. Xylanases are essential in ruminal digestion, where xylose monomers can be released to provide a nutritive source of non-xylanolytic ruminal bacteria.
Given the industrial significance of xylanases there is great interest to find new, highly active, inexpensive xylanases. These enzymes should be active at a wide range of pH and temperature, and they should function in both prokaryotic as well as eukaryotic expression systems.
“Xylanases are produced by a variety of microorganisms, and a number have been identified. Xylanase gene classifications can be divided into two main families: Clan GH?A (previously F family glycosylhydrlases), and Clan GH?C (previously G family glycosylhydrlases). (Henrissat et. al., 1989; Henrissat & Bairoch, 1993). Numerous microbial xylanases were identified in the literature. Fungal xylanases that originate from Aspergillus species (Arai and al. 1998, Ito et al. 1992), Emericella nidulans (MacCabe et al. 1996, previously identified as Aspergillus nidulans), and Penicillium chrysogenum (Haas et al. 1993) encode polypeptides with between 327- 347 amino acid. Fursariumoxysporum Xylanases (RuizRoldan and Sheppard, 1998; Sheppard and Sheppard, 1994) have a conserved catalytic and a cellulose binding and linker domains that attach the cellulose binding domain to their catalytic domains. The polypeptides have 384 amino acid with the addition domains. U.S. Pat. No. 5,763,254 to Woldike et al. This document discloses the DNA sequences, and derived amino acids sequences, of F.oxysporum C?family cellobiohydrolase and F.oxysporum F?family cellulase and F.oxysporum C?family endoglucanase. Each of these enzymes possess a carbohydrate binding region, a linker area, and a catalytic domain.
“The fungus Coniothyrium mintans Campbell can attack the fungal plant pathogen Sclerotinia sclerotiorum Lib (Huang & Hoes 1976), resulting the destruction of sclerotia and hyphae of the pathogen (Huang & Kokko 1987, 1988). Jones et al. (1974) reported endo and exo-1.3-?-glucanase activity on C. minitans. International application No. International application No. The prior art does not consider C. minitans to be a source of xylanase gene isolation.
“The invention provides a novel gene for xylanase (described herein as Cxy1) from the soil-borne fungus Coniothyrium mintans. SEQ ID NOS 1 and 2 show the DNA sequences of the cxy1 and Cxy1 genes, respectively. The cxy1 genome is compatible with both prokaryotic and eukaryotic expression systems. This makes it especially useful for industrial applications such as the improvement of nutrition availability of carbohydrates in animal feeds.
The following definitions will help you to understand the claims and specification.
“Catalytic domain” refers to the area of an enzyme’s amino acid sequence that contains the catalytic site.
“Carbohydrate binding domain” refers to an amino acid sequence that can bind the enzyme to a cellulose/hemicellulose substrate.
“Coding sequence” refers to the portion of a gene that codes for the amino acids sequence of a protein or for functional RNAs such as a trans- or rRNA.
“Complement” or “complementary sequence” refers to a sequence that forms a hydrogen-bonded doubleplex using another sequence of nucleotides following Watson-Crick base-pairing principles. The complementary base sequence for 5?-AAGGCT-3′ is 3′-TTCCGA-5″.
“Downstream” refers to any DNA or RNA site on the 3′ side.
“Expression” refers to transcription of a gene in structural RNA (rRNA), tRNA or messenger RNAs (mRNA), with subsequent translation into protein.
An amino acid sequence that is functionally equivalent to C. mintans Cxy1 can be modified by one or more amino acid substitutions or chemical modifications, but still retains the C. minitans Cxy1 xylanolytic activities. “Functionally equivalent” nucleotide sequences include those that encode polypeptides with substantially the same biological activity.
“Two nucleic acids sequences can be ‘heterologous’ if they are derived from different organisms.
“Hemicellulose” refers to glucans (apart form starch), mannans, or polyglucuronic, or polygalacturonic acids.
“A \”hemicellulose-degrading enzyme\” is an enzyme that catalyzes the degradation of hemicellulose.”
“Homology” refers to the degree of identity between two amino acid or nucleotide sequences.
“Isolated” refers to a substance or composition that has been removed from its natural state by man. A composition or substance that is ‘”isolated” occurs naturally in the environment. It has been altered or removed from its natural state, or both. A polynucleotide, or a protein, naturally found in living animals, is not “isolated”, but the same polynucleotide, or polypeptide, is separated from its coexisting material and is “isolated”, as this term is used herein.
“Linker region” refers to an amino acid sequence which operably links two functional domains within an enzyme.
“Nucleic Acid construct” refers to a nucleic acids molecule that has been isolated from a naturally occurring genetic gene, or modified to contain nucleic acids in segments that are combined and juxtaposed in ways not found in nature.
“Nucleic acid molecules” are single- or two-stranded, linear polynucleotides containing deoxyribonucleotides and ribonucleotides. They are linked by 3′-5?-phosphodiester bonds.
Two DNA sequences can be “operatively linked” if their nature does not affect the ability of each sequence to perform its normal functions. A promoter region could be linked to a sequence of coding if it was capable of triggering transcription.
A “polypeptide” is a linear polymer made of amino acids linked by peptide bonds.
“Promoter” is a cis-acting DNA sequence that is generally between 80 and 120 base pairs in length and located upstream from the initiation site for a gene. RNA polymerase can bind to it and initiate transcription.
“A “recombinant? nucleic acids molecule is, for example, a recombinant nucleic acids molecule. It is a new nucleic sequence that has been formed in vitro by the ligation two or more nonhomologous nucleic acids molecules (for instance, a recombinantplasmid containing one to more inserts of foreign genetic DNA cloned into the polylinker or its cloning site).
“Transformation” refers to the direct modification of a cell’s genome by externally applying purified recombinant genetic material from another cell. This results in the cell’s uptake and integration of the DNA. Recombinant DNA in bacteria is not integrated into the bacterial genome, but rather replicates itself as a “plasmid”.
“Upstream” refers to the 5′ side at any DNA or RNA site.
A vector is a nucleic acids molecule that can replicate autonomously in a host cells and accept foreign DNA. Each vector has its own origin of reproduction, one or more unique recognition spots for restriction endonucleases that can be used to insert foreign DNA. There are also selectable markers like genes coding for antibiotic resistance and recognition sequences (e.g. The promoter is used to express the inserted DNA. Common vectors are plasmid vectors or phage vectors.
In the context of a polypeptide “Xylanolytic Activity” refers to the ability of the polypeptides to release xylose from a substrate containing xylan.
“The invention is a novel xylanase genome obtained from Coniothyrium mintans. It is denoted as cxy1. The sequence of nucleotides for the cxy1 genes is described in SEQ ID No: 1. C. minitans Cxy1 encodes a xylanolytic polypeptide. SEQ ID NO. 2 shows the amino acid sequence for Cxy1.
“Those skilled in the art will appreciate the fact that many functionally identical nucleotide sequences encode a single amino acid sequence due to degeneracy of genetic code. The invention includes all nucleotide sequences which encode the Cxy1 sequence of xylanase, as shown in SEQ ID No: 2.
“Further C. minitans strains may contain naturally occurring variants of cxy1 genes that encode variants Cxy1 enzymes with xylanolytic activities that are substantially the same as the Cxy1 sequence shown in SEQ ID No: 2. The invention covers all such allelic variants and the encoded Cxy1xylanase.
“Using the techniques detailed in the Examples, the cxy1 sequence shown in SEQ ID No: 1 can be used for designing primers (such the Ff1/Fr5 prime pair described in these Examples herein) to amplify homologous sequences of C. minitans and other organisms using polymerase chain reactions (PCR). Or for the construction labeled probes (e.g. For use in nucleic acids hybridization assays to identify homologous sequences, biotin-labeled and radio-labeled These sequences can then been tested using the Examples herein to determine if they are capable of expressing polypeptides with xylanolytic activities. These methods allow those skilled in the art to identify variants of the cxy1xylanase genes or nucleotide sequences which encode polypeptides with xylanolytic activities.
“Additionally, skilled persons in the art can use standard mutagenesis techniques in conjunction with the assays for xylanolytic activities described in the Examples to obtain altered cxy1 genes and test them for expression of polypeptides that have xylanolytic ability. Useful mutagenesis techniques known in the art include, without limitation, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (see e.g. Sambrook et al., 1989 and Ausubel et al., 1999).”
“In order to obtain variant cxy1 sequences, those with ordinary skill in art will recognize that proteins can be modified by certain amino acids substitutions, additions and deletions without losing or reducing biological activity. It is also well-known that amino acid substitutions (or substitutions of one amino acids for another of the same size, charge and polarity) are unlikely to alter the function of proteins. There are 20 standard amino acids which make up proteins. They can be divided into four groups: The nonpolar (hydrophobic), group includes alanine (isoleucine), leucine (phenylalanine), proline (tryptophan), valine, tryptophan, proline and tryptophan); the polar (uncharged and neutral) group includes asparagine (glycine), cysteine and glutamine; the positively charged, basic (basic) group has lysine, histidine, aspartic acid (acidic acid and glutamic). It is unlikely that a substitution of an amino acid within a protein will have an adverse impact on its biological activity.
“As shown at FIGS. FIGS. 3A and 3B show that xylanaseCxy1 has the greatest homology with other F-family F-xylanases in the catalytic domain. In accordance with natural selection principles, it is well-known that amino acids and sequences of amino acid that are essential for the biological activity of a protein are tightly conserved among related proteins. The artifacts of substitutions, additions deletions and modifications to amino acids in the Cxy1 sequence will have a lower likelihood of affecting the enzyme’s xylanolytic activity than equivalent changes made within highly conserved regions. It is therefore expected that modifications, additions, deletions and substitutions of amino acids within the Cxy1 sequence would have the least impact on the enzyme’s xylanolytic activity if they occurred in a region with little or no conservation.
“In light of the above, nucleotide sequencings with at least 80 percent homology, more preferably at minimum 85% homology and more preferably a minimum of 95% homology with cxy1 sequence, which encodes polypeptides with xylanolytic activities, are covered by this invention. Amino acid sequences with at least 85%, more preferred at least 90% and more preferably a minimum of 95% homology to the Cxy1 sequence, which has xylanolytic enzyme sequence, which contains xylanase activity, which are included in SEQID NO: These homology scores are calculated by comparing the entire lengths of the two sequences that encode a polypeptide at either the amino acid or the DNA level. In a first embodiment, an isolated nucleic acids molecule is provided that encodes a polypeptide with xylanolytic activities. This encoded polypeptide comprises the sequence of amino acids shown in SEQ ID No: 2, or a functionally identical sequence with at least 85% homology.
“Cxy1’s xylanase structure is typical for F-family fungal species, as discussed in Example 4 (see below). Cxy1 also contains carbohydrate binding domains and linker domains. These structures have been found in F-family fungal xylanases such as F. oxysporum and Trichoderma reesei and Phanerochaete chrysosporium. Analysis of the sequence suggests that the portion of SEQ ID No: 2 spanning amino acids 1 through 17 has the characteristics of a signal protein (Nielsen and al., 1997). The xylanolytic activity may be affected by the deletion of the Cxy1 sequence, which runs from amino acids 1 through 22. However, it is not likely that this will affect the enzyme’s target. In another embodiment, the invention provides an isolate nucleic acid molecule that encodes a polypeptide with xylanolytic activities. This polypeptide comprises the amino acid sequence shown in SEQ ID No: 2 from amino acids 22 through amino acid 384 or a functionally identical sequence with at least 85% homology.
“The SEQ ID No: 2 segment from amino acids 23 through 52 is a sequence characteristic of a carbohydrate binding domain (“CBD \”),) which functions to tether cellulolytic and hemicellulolytic enzymes onto a cellulose/hemicellulose substrate. The SEQ ID NO. 2 segment that runs from amino acid 53 to amino acid 83 is a sequence which is characteristic of a linker, or hinge region. It may be used in coordination of the teriary structures of the carbohydrate binding domain and the catalyticdomain during xylan catalysis.
“Catalytic domain includes amino acids 84-384, of SEQ ID No: 2. Numerous regions of homology in the Cxy1 catalyticdomain are found with the catalytic domains for xylanases of fungal organisms such as Penicillium and Aspergillus (see FIGS. 3A-3E. The 17 amino acids located at positions 343-359 share 88-100% identity with the other xylanases. The 14 amino acids located at positions 242-255 share 93-100% identity (13 to 14 identical amino acids) and the other xylanases. The 11 amino acids located at positions 124-134 share 91-100% identity (10, or 11 identical amino acid) with the other depicted xylanases.
“The enzymatic hydrolysis (or hydrolysis) of the glycosidic bonds occurs at the active site of an xylanase. This requires two crucial residues: a proton donors and a nucleophile/base. (Davies & Henrissat 1995). Two major mechanisms are involved in hydrolysis. They can either result in an inversion or overall retention of anomeric structure. Cxy1’s two amino acid residues that act as both the proton donor or the nucleophile/base are Glu 209 & Glu 320. Comparison to other xylanases can be made at http://expasy.hcuge.ch. FIGS. 3A-3E show discrete areas of nearly identical or identical conservations of amino acids around the two Glu residues. 3A-3E. 3A-3E.
These xylanases only contain a catalytic domain and don’t contain linker regions or carbohydrate binding domains. These other xylanases only have catalytic activity and there are no regions of homology to the Cxy1 Catalytic Domain. Therefore, it is expected that the Cxy1 domain would display xylanolytic activity without the linker and carbohydrate binding domains. In a further embodiment, an isolated nucleic acid molecular is provided that encodes a polypeptide with xylanolytic activities. The polypeptide comprises the sequence of amino acids shown in SEQ ID No: 2, from amino acid 84 through amino acid 384, or a functionally identical sequence with at least 85% homology.
“The stability and activity of Cxy1’s catalytic domain will be enhanced by the carbohydrate binding region and linker area of Cxy1 (amino acid 23-83 of SEQ ID No: 2) It is believed they would also enhance the stability and activity of other xylanase catalytic domains, or those of other hemicellulose-degrading enzymes, if operably linked to such catalytic domains. In another embodiment, the invention covers a nucleic acids molecule that encodes the Cxy1 carbohydrate binding domain and linker area (the sequence of amino acids shown in SEQ ID No: 2, from amino acid 23 through amino acid 83), as well as a functionally identical sequence with at least 80% homology to it. The invention further extends to a nucleic acid construct comprising this nucleic acid molecule operably linked to a nucleic acid molecule that encodes a catalytic domain of a hemicellulose-degrading enzyme and that is heterologous to this nucleic acid molecule.”
“Cxy1 xylanase is very important for biotechnological and agricultural applications. This is because it has a functional compatibility with E. coli’s prokaryotic expression system (see Example 5). C. minitans, a higher-eukaryotic fungus that belongs to the Basidiomycota or Ascomycota subphylas, depends on its reproductive pattern. The cxy1 gene will likely be compatible with higher eukaryotic system (eukaryotic plant, yeast expression systems), for mass production and as a potential resistance gene to phytopathogens like S. sclerotiorum. In Example 6, cxy1 is expressed in Pichea pastoris, an eukaryotic organism. Functional compatibility of cxy1 and microbial systems is important for ruminal biotechnology. Transferring the cxy1 cDNA to non-xylanolytic ruminal bacterias or lower ruminal fungi is important. Additionally, ruminal biotechnology can improve the efficiency of xylan rich forage fiber digestion in ruminant livestock by overexpressing cxy1. Further embodiments of the invention include cells other than C.minitans that have been transformed with a nucleic acids molecule encoding C.minitans xylanase Cxy1 and a variant thereof, and methods for creating a polypeptide with xylanolytic activities. These methods include cultivating such cells in conditions that promote the expression and then recovering the encoded peptide from the culture. The invention extends to vectors that contain nucleic acids molecules of the invention that encode polypeptides with xylanolytic activities. These vectors usually contain at least one promoter signal and a termination signal for transcription.
For the recombinant production xylanase Axy1, industrial strains of microorganisms may be used (e.g. Aspergillus, Aspergillus ficuum or Aspergillus oryzae), or species of plants (e.g. canola or soybean, corn, potato or barley). The heterologous expression (or heterologous expression) of xylanase cxy1 begins with the assembly of an expression construct that includes the cxy1 sequence and control sequences like enhancers, promoters, and terminators. You may also include signal sequences or select markers. A secretory signal sequence may be included in the expression construct to achieve extracellular expression. If cytoplasmic expression is desired, the signal sequence may not be included in the expression construct. Both the promoter sequence and the signal sequence are essential for the expression and secretion Cxy1 proteins in host cells. To ensure transcription is efficient, transcriptional terminators may be included. The expression construct may include ancillary sequences that enhance expression or purify protein.
According to the invention, “Variable promoters (transcriptional initiating regulatory region)” may be used. The proposed expression host will determine the choice of the appropriate promoter. The native promoter that is associated with the C. minitans cxy1 gene may be used as the promoter. Promoters from other sources can also be used, provided they are functional in the host. E. coli tac, trc and Bacillus subtilis signal sequences (Brosius et. al. 1985), E. coli sacB promoters and signal sequence (Wong 1989), and oleosin specific promoters from Brassica napus and Arabidopsis thaliana. (van Hartingsveldt et. al. 1994).
“Promoter selection also depends on the desired efficiency of the peptide/protein production. To dramatically increase protein expression, inducers like aoxl and lac are used. The host cell may become damaged if too many proteins are expressed. Host cell growth could be restricted as a result. Inducible promoter systems allow host cells to grow to acceptable levels before inducing gene expression. This allows for higher product yields. The degree of homology to the promoter at the target locus may influence promoter selection if the protein coding sequence is to become integrated by a gene substitution (omega insert) event.
According to the invention, there may be many signal sequences. You can use a signal sequence that is homologous with the cxy1 sequence. A signal sequence that has been specifically selected or engineered to improve secretion in the expression host could also be used. B. subtilis’ sacB signal sequence is suitable for secretion in B. subtilis. Saccharomyces cerevisiae’s?-mating and P. pastoris acidphosphatase phoI signal signals are also suitable for P. pastoris secretion. If the protein coding sequence will be integrated via an omega insertion event, a signal sequence that is homologous to the target locus might be necessary. The signal sequence can be linked directly to the protein-coding sequence by linking the sequence encoding signal peptidase site to the signal sequence or using a nucleotide link consisting of fewer than ten codons.
“Elements that enhance transcription and translation have been identified in eukaryotic proteins expression systems. Placing the cauliflower mosaic virus (CaMV), promoter 1000 bp either side of a heterologous promotor may increase transcriptional levels by 10 to 400-fold. You should include appropriate translational initiation sequences in the expression construct. The expression construct can be modified to include the Kozak consensus sequencing for proper translational initiation. This may raise the level of translation 10 times.
The expression construct may contain elements that will enhance the purification of the protein. The product of oleosin fusions, for example, is a hybrid protein that contains the oleosin genes and the gene product of the interest. Van Hartingsveldt and al. 1994). To facilitate the purification of the recombinant oil body fusion proteins, it is possible to exploit their association (van Hartingsveldt and al. 1994). You can use vectors with a polyhistidine label. Because it binds to divalent cations like Ni2+, the polyhistidine tag makes it easier to purify the expressed polypeptide using known metal affinitychromatography protocols.
“A selection marker, which can be part of an expression construct or separately (e.g. carried by the expression vector), is used to integrate at different sites from the gene of concern. These markers can confer resistance against antibiotics (e.g. bla confers resistance for E.coli host cells to ampicillin, while npII confers resistance to B. napus cell lines to kanamycin) or allow the host to grow on minimal media (e.g. HIS4 allows P. pastoris, GS 115 His, to grow in the absence histidine). To allow independent expression, the selectable marker will be able to have its own transcriptional or translational initiation/termination regulatory regions. Antibiotic resistance can be used as a marker. The concentration of antibiotics for selection will depend on the antibiotic. It is generally between 10 and 600 mg/mL.
“The expression construct can be assembled using known recombinantDNA techniques (Sambrook and Ausubel, 1989; Ausubel and al. 1999). The basic steps to join two DNA fragments are restriction enzyme digestion and ligation. Modifications to the ends of the DNA fragment might be necessary before ligation. This could include filling in overhangs or deleting the terminal parts of the fragment(s), using nucleases (e.g. ExoIII), site-directed mutagenesis, or adding base pairs via PCR. To facilitate the joining of certain fragments, adaptors and polylinkers may be used. The expression construct is usually assembled using rounds of restriction, ligation and transformation of E. coli. There are many cloning vectors that can be used to construct the expression construct. The gene transfer system used to introduce the expression construct into the host cells will influence the choice of cloning vector. The resulting construct can be examined at the end of each stage by restriction, DNA sequence and hybridization, as well as PCR analysis.
The expression construct can be used to transform the host into a cloning vector construct. It may also be removed from the vector and used as is or transferred onto a delivery vector. The delivery vector allows for the maintenance and introduction of the expression construct in the chosen host cell type. The expression construct can be introduced into host cells using any one of the many known gene transfer methods (Ausubel and Sambrook, 1999; Sambrook and al. 1989). The host cells and the vector systems used will determine which gene transfer system is chosen.
“For example, Pichea pastoris cells can be introduced to the expression construct by electroporation or protoplast transformation. The conversion efficiencies of electroporation for P. pastoris are comparable to those achieved by spheroplasts. P. pastoris cells can be washed in sterile water, and then resuspended with a low conductivity solution (e.g. 1 Msorbitol solution). Transient pores are created in the cell membrane by a high voltage shock to the suspension. This allows the transforming DNA (e.g. expression construct) to enter the cells. Integration of the expression construct into the aoxl locus (alcoholoxidase) ensures that it is stable.
“Alternatively, an expression construct that contains the sacB promoter, signal sequence operably linked with the protein coding sequence is carried on pUB110. This plasmid can autonomously replicate in B. subtilis cell. Transform the plasmid construct to B. subtilis cells. Bacillus subtilis cells develop natural competence (i.e. When grown in nutrient-poor environments, the cells of Bacillus subtilis are able to absorb and integrate foreign DNA.
“In a further alternative, relating to higher plants, Brassica napus cells are transformed by Agrobacterium-mediated transformation. The expression construct is placed onto a binary vector that can be replicated in Agrobacterium Tumefaciens. This allows for mobilization of the construct into plant cells. The resultant construct is then transformed into A. Tumefaciens cells with an attenuated Ti, or a “helper virus”. The expression construct is then transferred to B. napus cells via conjugal mobilization using the binary vector:-expression construct. The expression construct is integrated at random into the plant’s genome.
“Host cells containing the expression construct (i.e. transformed cells) can be identified by using the selectable marker carried in the expression construct/vector and confirmed presence of the gene of concern by a variety techniques, including nucleic acids hybridization, PCR and antibodies. Transformed plant cells can be grown into whole plants or varietal lines of transgenic crops by screening.
“Transformed microbial cell can be grown using a variety of techniques, including batch or continuous fermentation on liquid media (Gerhardt and al. 1994). Transformed cells can be propagated in conditions that maximize product-to-cost ratios. You can increase product yields by manipulating cultivation parameters like temperature, pH, and media composition. The cultivation conditions for E.coli cells recombinantly hyper-expressing E.coli may be controlled and monitored carefully to ensure that the culture yields and biomass are at least 150 g (wet) and 5 g, respectively. To reduce the proteolysis of an over-expressed protein or peptide, low concentrations of a protease inhibitor (e.g., pepstatin or phenylmethylsulfonyl fluoride) can be used. To reduce or eliminate the degradation of the desired protein, host cells deficient in protease may also be used.
“Following fermentation, the microbes can be removed from the medium by known downstream processes like centrifugation or filtration. The nutrient media can be removed from the medium if the desired product is produced. Intracellular production involves the extraction of the desired product from the nutrient medium by breaking down cells using mechanical forces, ultrasound, chemicals, and/or high-pressure. To facilitate product purification, it is possible to produce an insoluble product such as in hyper-expressing E.coli systems. You can extract the product inclusions from damaged cells using centrifugation. Contaminating proteins can be washed with a buffer containing low levels of a denaturant (e.g. 0.5 to 6M urea, 0.1% sodium dodecyl-sulfate, or 0.5 to 4.05 M guanidine HCl). You can dissolve the washed inclusions in solutions that contain 6-8 M urea, 1 – 2% sodium decyl sulfate, or 4 to 6 m guanidine HCl. You can renature the solubilized product by slowly removing any denaturing agents during dialysis.
“Various methods may be used to purify the product from microbial ferment. These methods include precipitation (e.g. ammonium sulfurate precipitation), chromatography, gel filtration, ion exchanging, affinity liquid chromatography), ultrafiltration and electrophoresis.
“It will be obvious to those with ordinary skill in art that alternative methods and reagents, procedures, and techniques can be used or easily adapted to the practice of this invention. These examples are not intended to limit the invention. All abbreviations herein are the standard abbreviations in the art. The art is well-versed in specific procedures that are not detailed in the Examples.
“EXAMPLE 1”
“Cloning and Characterizing Genomic XylanaseDNA Fragment From Coniothyrium mintans”
“Genomic DNA Isolation From Coniothyrium Minitans Strain RRS 2134”
“Primer Design for the Polymerase Chain Reaction Amplification (PCR) of a Genomic C. Minitans Fragment”
“Xylanase amino acid sequences from fungal species Fursarium oxysporum, Aspergillus aculeatus, (Sheppard et. al. 1994), Emericella nidulans, MacCabe et. al. (1996), Humicola grisea, (likura et. al. 1997) were aligned in order to search for regions of homology. These four fungi are divergent species. There were two regions that showed homology. Rather than utilizing the amino acid sequence to then deduce the DNA homology, the DNA sequences corresponding to these two regions of homology were directly compared to generate semi-degenerate primers Ff1 (5′ gagaa(tc)agcatgaa(ag)tggga(tc)gc 3′) SEQ ID NO: 3 and Fr5 (5′ gtc(ag)g(act)(gac)ac(ta)ccccagac(ga)gt 3′) SEQ ID NO: 5 in order to amplify a homologous xylanase fragment from C. minitans genomic DNA by PCR (FIG. 1). The Ff1 and Fr5 primer pairs showed high levels of conservation at the nucleotide-level. There were no nucleotide ambiguities at the third nucleotide of either AG (which are common ambiguities caused by transition mutations during evolutionary evolution) or TC (which are common ambiguities). For Fr5, A/T and T substitutions were also found, while some substitutions weren’t taken into account when designing primers. This resulted in the Ff1/Fr5 primer pairing with high specificity for the target C. mintans xylanase sequence, without any high ambiguity.
“PCR Amplification and Cloning of the Genomic C. Minitans Xylanase Like Fragment, Ffaml
“PCR reactions were visualized under ultraviolet light and resolved using a 1.5% Agarose gel. Semi-purification was achieved by passing through a 1ml SEPHADEX DNA grade M (Pharmacia product no. 170045-01) column, and then ligated overnight into a pGEMT PCR vector system (Promega Corporation at 2800 Wood Hollow Road Madison, WI, 53711-5399 USA, product number A3600). The ligation mix was transformed into MAX EFFICIENCY DH5?COMPETENT CELLS (Life Technologies, product #18258-012) and recombinant vectors were screened by blue-white/ampicillin selection (white LacZ- cells are indicative of recombinant clones, transformants are selected through ampicillin resistance). The recombinant clones’ plasmid DNA (pDNA), was extracted using the WIZARD PLUS minipreppurification system (Promega Corporation product #18258-012) and sequenced using the universal 24-base M13 forward (5? cgc ggt ccc agt gac 3?) and the 24-base reverse (5? agc gga taa tt cac aca 3) sequencing primers with a fluorescent dye-terminator reaction kit, PE Applied Biosystems, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster Centre Drive, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster Center Drive, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, USA, Applied Biosystems (ABI-TERMINATOR SEQUENCING READYE 93, California, USA, 944, USA, Applied Biosystems (850 Lincoln Centre Drive), Foster City, Foster City, CA, 94404 USA, Applied Biosystems (944)
“Characterizing C. Minitans Xylanaselike Genomic Fragment Sequence.”
“The sequence of C. minitans 693 bp genome fragment was extracted from the sequenced pGEM?T clone. It was then entered into an alignment search algorithm, BLAST, Altschul et al. 1997) to find homologous sequences. C. minitans’ genomic fragment was PCR-tested and showed high homology with F-family fungal xylanases originating in A. aculearus (likura et. al. 1998; MacCabe et. al. 1996; RuizRoldan et. al. 1998; Sheppard et. al. 1994).
“EXAMPLE 2”
“Assessing the Presence, Size, and Abundance of a C. Minitans Xylanase Hybridizing Transcript
“RNA isolation from C. Minitans strains A11-3B 2A2, 2134, and A10-4”
“Northern Hybridization Analysis for Total C. Minitans’ RNA”
“Ten micrograms of total RNA was loaded and resolved on a 1.5% agarose gel containing 1.9% formaldehyde, and 0.02M MOPS (3-(N-morpholino)propane sulfonic acid) buffer. The RNA was transferred onto a nylon membrane (MAXIMUM STRENGTHNYTRAN, Schleicher & Schuell PO Box 2012 Keene N.H. 03431, USA product #77404) using Northern transfer (Sambrook et. al. 1989). It was then immobilized by UV crosslinking.
The resulting autoradiogram showed significant expression of a 1.3 kb transcription which hybridized with the C. mintans genomic xylanase fragment. This indicates that C. minitans strain C11-3B 2A2 had abundant expression of a xylanase like transcript in the presence ground sclerotia (S. sclerotiorum) as the sole source of carbohydrate. The xylanase like transcript was also found in total RNA from wild-type strain 2134, but it was less prevalent in strain A10-4 grown in minimal Czepek?Dox medium with ground sclerotia S. sclerotiorum. C. minitans strains 2134 (wild type) M11-3B2A2 were not able to produce a xylanase like transcript.
“EXAMPLE 3”
“Constructing and Screening a C. mintans cDNA library in the Isolation and Sequencing the Full-length Xylanase Transcript Sequencing (cxy1)”
“C. minitans strain 2A2 was grown in Czepek-Dox broth media that contained 1% ground Sclerotia of S.clerotiorum. It was kept for 15 days. As in Example 2, mycelia was taken and ground in liquid nitrogen. TRIZOL solution (Life Technologies) was used to extract total RNA. The total RNA mixture was then purified using a cellulose bound oligo-dT purification device (MESSAGEMAKER, Life Technologies product #10551-018). First strand cDNA was synthesized from the purified mRNA using SUPERSCRIPT II RNAse H reverse-transcriptase (Life Technologies, product #18053-017) and was cloned unidirectionally into a ?ZAP-cDNA GIGAPACK III GOLD cloning kit (Stratagene product #200450). Bacteriophage clones are packaged with?phage GIGAPACKIII GOLD packaging extract (Stratagene product #200450). They were then titered onto NZY media (1% (w/v), NZ amine 0.5% (w/v), NaCl 0.5% (w/v), 0.5% (w/v), yeast extract (Bacto), 0.2%(w/v), MgSO4.7H2 0. pH 7.0). On 15 cm Petri plates, phages were plated at a density 50,000 per plate.
“EXAMPLE 4”
“Characterization of cxy1 DNA by Amino Acid Sequence Alignment: Characterization and Structural Analysis”
“Phylogenetically, fungal species can be quite different in terms of their non-functional genomic sequences. However, functional sequences that are required for specific biological functions (such as catalytic or functional domains of cellulolytic enzymes) are often high in conservation. Comparing amino-acid sequences with other fungal xylanases may help identify functional domains within Cxy1.
Cxy1 seems to have a similar structural layout to many F-family fungal Fxylanases. Typical fungal xylanases, such as the Aspergillus kawachii xylanase XynA, (Ito el., 1992), and the Emericella nidulans xylanase XylnC, (MacCabe et., 1996), have a unique Nterminus that is followed by a catalytic domain at the C-terminus. Cxy1 seems to have extensive homology in the C-terminus catalytic area. However, Cxy1’s xylanase may also include a carbohydrate binding domain (CBD). This domain is located between amino acids 22 and 52. It is flanked by WSQCGG and WYFQC (SEQID NO: 2, amino acid 22-27) and WYFQC motifs. These sequences are very similar to those of microbial glycanases (Gilkes et. al. 1991). The conservation of the entire sequence suggests that it may still have carbohydrate binding function. The CBD domain is used in cellulolytic and hemicellulolytic enzymes to tether enzyme to a substrate. This enhances substrate specificity, as well as degradative activity. However, the presence of a CBD-like sequence does not necessarily mean that an enzyme will exhibit cellulolytic activities. For instance, the Pseudomonas fluorescens xylanase Xyna contains a cellulose-binding domain, but does not demonstrate hydrolytic activity towards carboxymethylcellulose (CMC)(Gilbert et al., 1988; Hall et al., 1989). Some xylanases have dual cellulolytic/xylanolytic functions. Clostridium thermocellurn F family xylanase, XynZ, exhibits xylanase activation with high specificity but low cellulase activity (Greprinet and al., 1988).
“The Cxy1 CBD has been linked to the Cterminus catalyticdomain by a unique linker sequence (SEQ ID No:2, amino acids 53 through 83), which may play a role in coordination of the tertiary CBD and catalytic domain during the xylan catalysis process (FIGS. 2A, 2B). This structural arrangement is very similar to the F-family xylanase homologues Ffam1 & Xyl3 found within the fungal phytopathogen Fursarium oxysporum. (Sheppard et. al. 1994; Ruiz?Roldan et. al. 1998). Cxy1 shows high homology with all F-family xylanases. The catalytic Cterminal domain (FIGS) is the most homological. 3A, 3B). Sequence conservation does not only apply to fungal F family cellulases but also extends to prokaryotic F.family xylanases like the thermostable xylanase XylA, which is found in the eubacteria Thermomonospora Alba (Blanco et. al., 1997) and the C. thermocellum XynY, (Fontes et. al., 1995).
“EXAMPLE 5”
“Assaying Cxy1 activity in Escherichiacoli”
Many eukaryotic enzymes need extensive post-translational modifications to maintain their functional properties. Functional reconstitution of eukaryotic protein in prokaryotic systems is often blocked by post-translational processing mechanisms like glycosylation (additions of glycosyl group to thr/ser(O-linked), or asn(N-linked). residues)) and proteolytic destruction. A cxy1-cDNA-llacZ fusion produced a significant amount of functional xylanase in Escherichia Coli. This suggests that Cxy1 activity is not dependent on post-translational modifications. It also indicates that Cxy1 can be used in conjunction with prokaryotic expression systems.
“Sequence Verification for the lacZ/cxy1fusion Clone”
“Several pBLUESCRIPT-cxy1 clones were created by removing the phagemid from a lambda vector from the C. mintans M11-3B2A2 cDNA database. To determine if the cxy1 insert would transcribe into a translated Cxy1 product, we sequenced pBLUESCRIPT cxy1 cells.
“Assaying the Xylanolytic Activity for the Cxy1/LacZ Fusion protein from E. Coli [strain DH5?]”
Summary for “Coniothyrium minitans xylanase gene Cxy1”
Cell wall structure and rigidity are achieved by the presence of cellulose, hempicellulose, and other lignin polymers. Complex carbohydrates are essential to maintain physical integrity and prevent plant pathogens from infecting your cells. Xylan is a polymeric chain made up of xylose carbohydrate monomers linked with successive?-1.4 xylose linkages. It makes up a large portion of plant hemicellulose. Xylan is responsible for the strength of plant secondary walls and limits the access of cellulases to cells wall. This prevents plant surface damage from plant pathogens.
“The hydrolysis of xylan is a key step in the degradation of plant tissues. This enzyme is produced by fibrolytic bacteria or fungi. A consortium of hydrolytic enzymes is responsible for xylan’s complex physical structure. Endoxylanlolytic enzymes (EC 3.2.1.8) break down xylan polymers into shorter oligoxylan pieces. The resulting oligoxylan bits are then degraded by?xylosidase (EC 3.2.1.37) (Bieley 1985). The hydrolysis of xylan can be complicated by various substituents, such as acetyl and arabinosyl along the elongated,?-1.4 xylose chain. The presence of different substituent groups in xylan polymers can vary between plant types. Xylan from grasses appears to be dominated by arabinose constituents. However, xylan polymers from hardwoods have acetyl- and arabinose substitutes. Endoxylanolytic hydrolysis is impeded by the steric impediment caused by these substituents attached the the?-1.4 xylan backbone. Xylanolytic degradation is therefore effected by xylanases in concert with enzymes such as ?-L-arabinofuranosidases (EC 3.2.1.55) and acetylesterases (EC 3.1.1.6), which cleave substituents from the xylan polymer.”
There are many applications for xylanolytic enzymes in biotechnology and industry. These include the addition of xylanases to biopulping processes. The enzymatic exclusion of xylan tissue from woody plants may improve pulp quality. Cellulases and/or xylanases can be used to improve the nutritional availability of feed carbohydrate and fermentative composting by predigesting forage crops. Xylanases can also be used to reduce organic waste disposal at landfill sites. Xylanases can also be used to improve quality of flour for baked goods. Xylan is a significant reserve of nutritive sugar in plant biomass. Xylanases are essential in ruminal digestion, where xylose monomers can be released to provide a nutritive source of non-xylanolytic ruminal bacteria.
Given the industrial significance of xylanases there is great interest to find new, highly active, inexpensive xylanases. These enzymes should be active at a wide range of pH and temperature, and they should function in both prokaryotic as well as eukaryotic expression systems.
“Xylanases are produced by a variety of microorganisms, and a number have been identified. Xylanase gene classifications can be divided into two main families: Clan GH?A (previously F family glycosylhydrlases), and Clan GH?C (previously G family glycosylhydrlases). (Henrissat et. al., 1989; Henrissat & Bairoch, 1993). Numerous microbial xylanases were identified in the literature. Fungal xylanases that originate from Aspergillus species (Arai and al. 1998, Ito et al. 1992), Emericella nidulans (MacCabe et al. 1996, previously identified as Aspergillus nidulans), and Penicillium chrysogenum (Haas et al. 1993) encode polypeptides with between 327- 347 amino acid. Fursariumoxysporum Xylanases (RuizRoldan and Sheppard, 1998; Sheppard and Sheppard, 1994) have a conserved catalytic and a cellulose binding and linker domains that attach the cellulose binding domain to their catalytic domains. The polypeptides have 384 amino acid with the addition domains. U.S. Pat. No. 5,763,254 to Woldike et al. This document discloses the DNA sequences, and derived amino acids sequences, of F.oxysporum C?family cellobiohydrolase and F.oxysporum F?family cellulase and F.oxysporum C?family endoglucanase. Each of these enzymes possess a carbohydrate binding region, a linker area, and a catalytic domain.
“The fungus Coniothyrium mintans Campbell can attack the fungal plant pathogen Sclerotinia sclerotiorum Lib (Huang & Hoes 1976), resulting the destruction of sclerotia and hyphae of the pathogen (Huang & Kokko 1987, 1988). Jones et al. (1974) reported endo and exo-1.3-?-glucanase activity on C. minitans. International application No. International application No. The prior art does not consider C. minitans to be a source of xylanase gene isolation.
“The invention provides a novel gene for xylanase (described herein as Cxy1) from the soil-borne fungus Coniothyrium mintans. SEQ ID NOS 1 and 2 show the DNA sequences of the cxy1 and Cxy1 genes, respectively. The cxy1 genome is compatible with both prokaryotic and eukaryotic expression systems. This makes it especially useful for industrial applications such as the improvement of nutrition availability of carbohydrates in animal feeds.
The following definitions will help you to understand the claims and specification.
“Catalytic domain” refers to the area of an enzyme’s amino acid sequence that contains the catalytic site.
“Carbohydrate binding domain” refers to an amino acid sequence that can bind the enzyme to a cellulose/hemicellulose substrate.
“Coding sequence” refers to the portion of a gene that codes for the amino acids sequence of a protein or for functional RNAs such as a trans- or rRNA.
“Complement” or “complementary sequence” refers to a sequence that forms a hydrogen-bonded doubleplex using another sequence of nucleotides following Watson-Crick base-pairing principles. The complementary base sequence for 5?-AAGGCT-3′ is 3′-TTCCGA-5″.
“Downstream” refers to any DNA or RNA site on the 3′ side.
“Expression” refers to transcription of a gene in structural RNA (rRNA), tRNA or messenger RNAs (mRNA), with subsequent translation into protein.
An amino acid sequence that is functionally equivalent to C. mintans Cxy1 can be modified by one or more amino acid substitutions or chemical modifications, but still retains the C. minitans Cxy1 xylanolytic activities. “Functionally equivalent” nucleotide sequences include those that encode polypeptides with substantially the same biological activity.
“Two nucleic acids sequences can be ‘heterologous’ if they are derived from different organisms.
“Hemicellulose” refers to glucans (apart form starch), mannans, or polyglucuronic, or polygalacturonic acids.
“A \”hemicellulose-degrading enzyme\” is an enzyme that catalyzes the degradation of hemicellulose.”
“Homology” refers to the degree of identity between two amino acid or nucleotide sequences.
“Isolated” refers to a substance or composition that has been removed from its natural state by man. A composition or substance that is ‘”isolated” occurs naturally in the environment. It has been altered or removed from its natural state, or both. A polynucleotide, or a protein, naturally found in living animals, is not “isolated”, but the same polynucleotide, or polypeptide, is separated from its coexisting material and is “isolated”, as this term is used herein.
“Linker region” refers to an amino acid sequence which operably links two functional domains within an enzyme.
“Nucleic Acid construct” refers to a nucleic acids molecule that has been isolated from a naturally occurring genetic gene, or modified to contain nucleic acids in segments that are combined and juxtaposed in ways not found in nature.
“Nucleic acid molecules” are single- or two-stranded, linear polynucleotides containing deoxyribonucleotides and ribonucleotides. They are linked by 3′-5?-phosphodiester bonds.
Two DNA sequences can be “operatively linked” if their nature does not affect the ability of each sequence to perform its normal functions. A promoter region could be linked to a sequence of coding if it was capable of triggering transcription.
A “polypeptide” is a linear polymer made of amino acids linked by peptide bonds.
“Promoter” is a cis-acting DNA sequence that is generally between 80 and 120 base pairs in length and located upstream from the initiation site for a gene. RNA polymerase can bind to it and initiate transcription.
“A “recombinant? nucleic acids molecule is, for example, a recombinant nucleic acids molecule. It is a new nucleic sequence that has been formed in vitro by the ligation two or more nonhomologous nucleic acids molecules (for instance, a recombinantplasmid containing one to more inserts of foreign genetic DNA cloned into the polylinker or its cloning site).
“Transformation” refers to the direct modification of a cell’s genome by externally applying purified recombinant genetic material from another cell. This results in the cell’s uptake and integration of the DNA. Recombinant DNA in bacteria is not integrated into the bacterial genome, but rather replicates itself as a “plasmid”.
“Upstream” refers to the 5′ side at any DNA or RNA site.
A vector is a nucleic acids molecule that can replicate autonomously in a host cells and accept foreign DNA. Each vector has its own origin of reproduction, one or more unique recognition spots for restriction endonucleases that can be used to insert foreign DNA. There are also selectable markers like genes coding for antibiotic resistance and recognition sequences (e.g. The promoter is used to express the inserted DNA. Common vectors are plasmid vectors or phage vectors.
In the context of a polypeptide “Xylanolytic Activity” refers to the ability of the polypeptides to release xylose from a substrate containing xylan.
“The invention is a novel xylanase genome obtained from Coniothyrium mintans. It is denoted as cxy1. The sequence of nucleotides for the cxy1 genes is described in SEQ ID No: 1. C. minitans Cxy1 encodes a xylanolytic polypeptide. SEQ ID NO. 2 shows the amino acid sequence for Cxy1.
“Those skilled in the art will appreciate the fact that many functionally identical nucleotide sequences encode a single amino acid sequence due to degeneracy of genetic code. The invention includes all nucleotide sequences which encode the Cxy1 sequence of xylanase, as shown in SEQ ID No: 2.
“Further C. minitans strains may contain naturally occurring variants of cxy1 genes that encode variants Cxy1 enzymes with xylanolytic activities that are substantially the same as the Cxy1 sequence shown in SEQ ID No: 2. The invention covers all such allelic variants and the encoded Cxy1xylanase.
“Using the techniques detailed in the Examples, the cxy1 sequence shown in SEQ ID No: 1 can be used for designing primers (such the Ff1/Fr5 prime pair described in these Examples herein) to amplify homologous sequences of C. minitans and other organisms using polymerase chain reactions (PCR). Or for the construction labeled probes (e.g. For use in nucleic acids hybridization assays to identify homologous sequences, biotin-labeled and radio-labeled These sequences can then been tested using the Examples herein to determine if they are capable of expressing polypeptides with xylanolytic activities. These methods allow those skilled in the art to identify variants of the cxy1xylanase genes or nucleotide sequences which encode polypeptides with xylanolytic activities.
“Additionally, skilled persons in the art can use standard mutagenesis techniques in conjunction with the assays for xylanolytic activities described in the Examples to obtain altered cxy1 genes and test them for expression of polypeptides that have xylanolytic ability. Useful mutagenesis techniques known in the art include, without limitation, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (see e.g. Sambrook et al., 1989 and Ausubel et al., 1999).”
“In order to obtain variant cxy1 sequences, those with ordinary skill in art will recognize that proteins can be modified by certain amino acids substitutions, additions and deletions without losing or reducing biological activity. It is also well-known that amino acid substitutions (or substitutions of one amino acids for another of the same size, charge and polarity) are unlikely to alter the function of proteins. There are 20 standard amino acids which make up proteins. They can be divided into four groups: The nonpolar (hydrophobic), group includes alanine (isoleucine), leucine (phenylalanine), proline (tryptophan), valine, tryptophan, proline and tryptophan); the polar (uncharged and neutral) group includes asparagine (glycine), cysteine and glutamine; the positively charged, basic (basic) group has lysine, histidine, aspartic acid (acidic acid and glutamic). It is unlikely that a substitution of an amino acid within a protein will have an adverse impact on its biological activity.
“As shown at FIGS. FIGS. 3A and 3B show that xylanaseCxy1 has the greatest homology with other F-family F-xylanases in the catalytic domain. In accordance with natural selection principles, it is well-known that amino acids and sequences of amino acid that are essential for the biological activity of a protein are tightly conserved among related proteins. The artifacts of substitutions, additions deletions and modifications to amino acids in the Cxy1 sequence will have a lower likelihood of affecting the enzyme’s xylanolytic activity than equivalent changes made within highly conserved regions. It is therefore expected that modifications, additions, deletions and substitutions of amino acids within the Cxy1 sequence would have the least impact on the enzyme’s xylanolytic activity if they occurred in a region with little or no conservation.
“In light of the above, nucleotide sequencings with at least 80 percent homology, more preferably at minimum 85% homology and more preferably a minimum of 95% homology with cxy1 sequence, which encodes polypeptides with xylanolytic activities, are covered by this invention. Amino acid sequences with at least 85%, more preferred at least 90% and more preferably a minimum of 95% homology to the Cxy1 sequence, which has xylanolytic enzyme sequence, which contains xylanase activity, which are included in SEQID NO: These homology scores are calculated by comparing the entire lengths of the two sequences that encode a polypeptide at either the amino acid or the DNA level. In a first embodiment, an isolated nucleic acids molecule is provided that encodes a polypeptide with xylanolytic activities. This encoded polypeptide comprises the sequence of amino acids shown in SEQ ID No: 2, or a functionally identical sequence with at least 85% homology.
“Cxy1’s xylanase structure is typical for F-family fungal species, as discussed in Example 4 (see below). Cxy1 also contains carbohydrate binding domains and linker domains. These structures have been found in F-family fungal xylanases such as F. oxysporum and Trichoderma reesei and Phanerochaete chrysosporium. Analysis of the sequence suggests that the portion of SEQ ID No: 2 spanning amino acids 1 through 17 has the characteristics of a signal protein (Nielsen and al., 1997). The xylanolytic activity may be affected by the deletion of the Cxy1 sequence, which runs from amino acids 1 through 22. However, it is not likely that this will affect the enzyme’s target. In another embodiment, the invention provides an isolate nucleic acid molecule that encodes a polypeptide with xylanolytic activities. This polypeptide comprises the amino acid sequence shown in SEQ ID No: 2 from amino acids 22 through amino acid 384 or a functionally identical sequence with at least 85% homology.
“The SEQ ID No: 2 segment from amino acids 23 through 52 is a sequence characteristic of a carbohydrate binding domain (“CBD \”),) which functions to tether cellulolytic and hemicellulolytic enzymes onto a cellulose/hemicellulose substrate. The SEQ ID NO. 2 segment that runs from amino acid 53 to amino acid 83 is a sequence which is characteristic of a linker, or hinge region. It may be used in coordination of the teriary structures of the carbohydrate binding domain and the catalyticdomain during xylan catalysis.
“Catalytic domain includes amino acids 84-384, of SEQ ID No: 2. Numerous regions of homology in the Cxy1 catalyticdomain are found with the catalytic domains for xylanases of fungal organisms such as Penicillium and Aspergillus (see FIGS. 3A-3E. The 17 amino acids located at positions 343-359 share 88-100% identity with the other xylanases. The 14 amino acids located at positions 242-255 share 93-100% identity (13 to 14 identical amino acids) and the other xylanases. The 11 amino acids located at positions 124-134 share 91-100% identity (10, or 11 identical amino acid) with the other depicted xylanases.
“The enzymatic hydrolysis (or hydrolysis) of the glycosidic bonds occurs at the active site of an xylanase. This requires two crucial residues: a proton donors and a nucleophile/base. (Davies & Henrissat 1995). Two major mechanisms are involved in hydrolysis. They can either result in an inversion or overall retention of anomeric structure. Cxy1’s two amino acid residues that act as both the proton donor or the nucleophile/base are Glu 209 & Glu 320. Comparison to other xylanases can be made at http://expasy.hcuge.ch. FIGS. 3A-3E show discrete areas of nearly identical or identical conservations of amino acids around the two Glu residues. 3A-3E. 3A-3E.
These xylanases only contain a catalytic domain and don’t contain linker regions or carbohydrate binding domains. These other xylanases only have catalytic activity and there are no regions of homology to the Cxy1 Catalytic Domain. Therefore, it is expected that the Cxy1 domain would display xylanolytic activity without the linker and carbohydrate binding domains. In a further embodiment, an isolated nucleic acid molecular is provided that encodes a polypeptide with xylanolytic activities. The polypeptide comprises the sequence of amino acids shown in SEQ ID No: 2, from amino acid 84 through amino acid 384, or a functionally identical sequence with at least 85% homology.
“The stability and activity of Cxy1’s catalytic domain will be enhanced by the carbohydrate binding region and linker area of Cxy1 (amino acid 23-83 of SEQ ID No: 2) It is believed they would also enhance the stability and activity of other xylanase catalytic domains, or those of other hemicellulose-degrading enzymes, if operably linked to such catalytic domains. In another embodiment, the invention covers a nucleic acids molecule that encodes the Cxy1 carbohydrate binding domain and linker area (the sequence of amino acids shown in SEQ ID No: 2, from amino acid 23 through amino acid 83), as well as a functionally identical sequence with at least 80% homology to it. The invention further extends to a nucleic acid construct comprising this nucleic acid molecule operably linked to a nucleic acid molecule that encodes a catalytic domain of a hemicellulose-degrading enzyme and that is heterologous to this nucleic acid molecule.”
“Cxy1 xylanase is very important for biotechnological and agricultural applications. This is because it has a functional compatibility with E. coli’s prokaryotic expression system (see Example 5). C. minitans, a higher-eukaryotic fungus that belongs to the Basidiomycota or Ascomycota subphylas, depends on its reproductive pattern. The cxy1 gene will likely be compatible with higher eukaryotic system (eukaryotic plant, yeast expression systems), for mass production and as a potential resistance gene to phytopathogens like S. sclerotiorum. In Example 6, cxy1 is expressed in Pichea pastoris, an eukaryotic organism. Functional compatibility of cxy1 and microbial systems is important for ruminal biotechnology. Transferring the cxy1 cDNA to non-xylanolytic ruminal bacterias or lower ruminal fungi is important. Additionally, ruminal biotechnology can improve the efficiency of xylan rich forage fiber digestion in ruminant livestock by overexpressing cxy1. Further embodiments of the invention include cells other than C.minitans that have been transformed with a nucleic acids molecule encoding C.minitans xylanase Cxy1 and a variant thereof, and methods for creating a polypeptide with xylanolytic activities. These methods include cultivating such cells in conditions that promote the expression and then recovering the encoded peptide from the culture. The invention extends to vectors that contain nucleic acids molecules of the invention that encode polypeptides with xylanolytic activities. These vectors usually contain at least one promoter signal and a termination signal for transcription.
For the recombinant production xylanase Axy1, industrial strains of microorganisms may be used (e.g. Aspergillus, Aspergillus ficuum or Aspergillus oryzae), or species of plants (e.g. canola or soybean, corn, potato or barley). The heterologous expression (or heterologous expression) of xylanase cxy1 begins with the assembly of an expression construct that includes the cxy1 sequence and control sequences like enhancers, promoters, and terminators. You may also include signal sequences or select markers. A secretory signal sequence may be included in the expression construct to achieve extracellular expression. If cytoplasmic expression is desired, the signal sequence may not be included in the expression construct. Both the promoter sequence and the signal sequence are essential for the expression and secretion Cxy1 proteins in host cells. To ensure transcription is efficient, transcriptional terminators may be included. The expression construct may include ancillary sequences that enhance expression or purify protein.
According to the invention, “Variable promoters (transcriptional initiating regulatory region)” may be used. The proposed expression host will determine the choice of the appropriate promoter. The native promoter that is associated with the C. minitans cxy1 gene may be used as the promoter. Promoters from other sources can also be used, provided they are functional in the host. E. coli tac, trc and Bacillus subtilis signal sequences (Brosius et. al. 1985), E. coli sacB promoters and signal sequence (Wong 1989), and oleosin specific promoters from Brassica napus and Arabidopsis thaliana. (van Hartingsveldt et. al. 1994).
“Promoter selection also depends on the desired efficiency of the peptide/protein production. To dramatically increase protein expression, inducers like aoxl and lac are used. The host cell may become damaged if too many proteins are expressed. Host cell growth could be restricted as a result. Inducible promoter systems allow host cells to grow to acceptable levels before inducing gene expression. This allows for higher product yields. The degree of homology to the promoter at the target locus may influence promoter selection if the protein coding sequence is to become integrated by a gene substitution (omega insert) event.
According to the invention, there may be many signal sequences. You can use a signal sequence that is homologous with the cxy1 sequence. A signal sequence that has been specifically selected or engineered to improve secretion in the expression host could also be used. B. subtilis’ sacB signal sequence is suitable for secretion in B. subtilis. Saccharomyces cerevisiae’s?-mating and P. pastoris acidphosphatase phoI signal signals are also suitable for P. pastoris secretion. If the protein coding sequence will be integrated via an omega insertion event, a signal sequence that is homologous to the target locus might be necessary. The signal sequence can be linked directly to the protein-coding sequence by linking the sequence encoding signal peptidase site to the signal sequence or using a nucleotide link consisting of fewer than ten codons.
“Elements that enhance transcription and translation have been identified in eukaryotic proteins expression systems. Placing the cauliflower mosaic virus (CaMV), promoter 1000 bp either side of a heterologous promotor may increase transcriptional levels by 10 to 400-fold. You should include appropriate translational initiation sequences in the expression construct. The expression construct can be modified to include the Kozak consensus sequencing for proper translational initiation. This may raise the level of translation 10 times.
The expression construct may contain elements that will enhance the purification of the protein. The product of oleosin fusions, for example, is a hybrid protein that contains the oleosin genes and the gene product of the interest. Van Hartingsveldt and al. 1994). To facilitate the purification of the recombinant oil body fusion proteins, it is possible to exploit their association (van Hartingsveldt and al. 1994). You can use vectors with a polyhistidine label. Because it binds to divalent cations like Ni2+, the polyhistidine tag makes it easier to purify the expressed polypeptide using known metal affinitychromatography protocols.
“A selection marker, which can be part of an expression construct or separately (e.g. carried by the expression vector), is used to integrate at different sites from the gene of concern. These markers can confer resistance against antibiotics (e.g. bla confers resistance for E.coli host cells to ampicillin, while npII confers resistance to B. napus cell lines to kanamycin) or allow the host to grow on minimal media (e.g. HIS4 allows P. pastoris, GS 115 His, to grow in the absence histidine). To allow independent expression, the selectable marker will be able to have its own transcriptional or translational initiation/termination regulatory regions. Antibiotic resistance can be used as a marker. The concentration of antibiotics for selection will depend on the antibiotic. It is generally between 10 and 600 mg/mL.
“The expression construct can be assembled using known recombinantDNA techniques (Sambrook and Ausubel, 1989; Ausubel and al. 1999). The basic steps to join two DNA fragments are restriction enzyme digestion and ligation. Modifications to the ends of the DNA fragment might be necessary before ligation. This could include filling in overhangs or deleting the terminal parts of the fragment(s), using nucleases (e.g. ExoIII), site-directed mutagenesis, or adding base pairs via PCR. To facilitate the joining of certain fragments, adaptors and polylinkers may be used. The expression construct is usually assembled using rounds of restriction, ligation and transformation of E. coli. There are many cloning vectors that can be used to construct the expression construct. The gene transfer system used to introduce the expression construct into the host cells will influence the choice of cloning vector. The resulting construct can be examined at the end of each stage by restriction, DNA sequence and hybridization, as well as PCR analysis.
The expression construct can be used to transform the host into a cloning vector construct. It may also be removed from the vector and used as is or transferred onto a delivery vector. The delivery vector allows for the maintenance and introduction of the expression construct in the chosen host cell type. The expression construct can be introduced into host cells using any one of the many known gene transfer methods (Ausubel and Sambrook, 1999; Sambrook and al. 1989). The host cells and the vector systems used will determine which gene transfer system is chosen.
“For example, Pichea pastoris cells can be introduced to the expression construct by electroporation or protoplast transformation. The conversion efficiencies of electroporation for P. pastoris are comparable to those achieved by spheroplasts. P. pastoris cells can be washed in sterile water, and then resuspended with a low conductivity solution (e.g. 1 Msorbitol solution). Transient pores are created in the cell membrane by a high voltage shock to the suspension. This allows the transforming DNA (e.g. expression construct) to enter the cells. Integration of the expression construct into the aoxl locus (alcoholoxidase) ensures that it is stable.
“Alternatively, an expression construct that contains the sacB promoter, signal sequence operably linked with the protein coding sequence is carried on pUB110. This plasmid can autonomously replicate in B. subtilis cell. Transform the plasmid construct to B. subtilis cells. Bacillus subtilis cells develop natural competence (i.e. When grown in nutrient-poor environments, the cells of Bacillus subtilis are able to absorb and integrate foreign DNA.
“In a further alternative, relating to higher plants, Brassica napus cells are transformed by Agrobacterium-mediated transformation. The expression construct is placed onto a binary vector that can be replicated in Agrobacterium Tumefaciens. This allows for mobilization of the construct into plant cells. The resultant construct is then transformed into A. Tumefaciens cells with an attenuated Ti, or a “helper virus”. The expression construct is then transferred to B. napus cells via conjugal mobilization using the binary vector:-expression construct. The expression construct is integrated at random into the plant’s genome.
“Host cells containing the expression construct (i.e. transformed cells) can be identified by using the selectable marker carried in the expression construct/vector and confirmed presence of the gene of concern by a variety techniques, including nucleic acids hybridization, PCR and antibodies. Transformed plant cells can be grown into whole plants or varietal lines of transgenic crops by screening.
“Transformed microbial cell can be grown using a variety of techniques, including batch or continuous fermentation on liquid media (Gerhardt and al. 1994). Transformed cells can be propagated in conditions that maximize product-to-cost ratios. You can increase product yields by manipulating cultivation parameters like temperature, pH, and media composition. The cultivation conditions for E.coli cells recombinantly hyper-expressing E.coli may be controlled and monitored carefully to ensure that the culture yields and biomass are at least 150 g (wet) and 5 g, respectively. To reduce the proteolysis of an over-expressed protein or peptide, low concentrations of a protease inhibitor (e.g., pepstatin or phenylmethylsulfonyl fluoride) can be used. To reduce or eliminate the degradation of the desired protein, host cells deficient in protease may also be used.
“Following fermentation, the microbes can be removed from the medium by known downstream processes like centrifugation or filtration. The nutrient media can be removed from the medium if the desired product is produced. Intracellular production involves the extraction of the desired product from the nutrient medium by breaking down cells using mechanical forces, ultrasound, chemicals, and/or high-pressure. To facilitate product purification, it is possible to produce an insoluble product such as in hyper-expressing E.coli systems. You can extract the product inclusions from damaged cells using centrifugation. Contaminating proteins can be washed with a buffer containing low levels of a denaturant (e.g. 0.5 to 6M urea, 0.1% sodium dodecyl-sulfate, or 0.5 to 4.05 M guanidine HCl). You can dissolve the washed inclusions in solutions that contain 6-8 M urea, 1 – 2% sodium decyl sulfate, or 4 to 6 m guanidine HCl. You can renature the solubilized product by slowly removing any denaturing agents during dialysis.
“Various methods may be used to purify the product from microbial ferment. These methods include precipitation (e.g. ammonium sulfurate precipitation), chromatography, gel filtration, ion exchanging, affinity liquid chromatography), ultrafiltration and electrophoresis.
“It will be obvious to those with ordinary skill in art that alternative methods and reagents, procedures, and techniques can be used or easily adapted to the practice of this invention. These examples are not intended to limit the invention. All abbreviations herein are the standard abbreviations in the art. The art is well-versed in specific procedures that are not detailed in the Examples.
“EXAMPLE 1”
“Cloning and Characterizing Genomic XylanaseDNA Fragment From Coniothyrium mintans”
“Genomic DNA Isolation From Coniothyrium Minitans Strain RRS 2134”
“Primer Design for the Polymerase Chain Reaction Amplification (PCR) of a Genomic C. Minitans Fragment”
“Xylanase amino acid sequences from fungal species Fursarium oxysporum, Aspergillus aculeatus, (Sheppard et. al. 1994), Emericella nidulans, MacCabe et. al. (1996), Humicola grisea, (likura et. al. 1997) were aligned in order to search for regions of homology. These four fungi are divergent species. There were two regions that showed homology. Rather than utilizing the amino acid sequence to then deduce the DNA homology, the DNA sequences corresponding to these two regions of homology were directly compared to generate semi-degenerate primers Ff1 (5′ gagaa(tc)agcatgaa(ag)tggga(tc)gc 3′) SEQ ID NO: 3 and Fr5 (5′ gtc(ag)g(act)(gac)ac(ta)ccccagac(ga)gt 3′) SEQ ID NO: 5 in order to amplify a homologous xylanase fragment from C. minitans genomic DNA by PCR (FIG. 1). The Ff1 and Fr5 primer pairs showed high levels of conservation at the nucleotide-level. There were no nucleotide ambiguities at the third nucleotide of either AG (which are common ambiguities caused by transition mutations during evolutionary evolution) or TC (which are common ambiguities). For Fr5, A/T and T substitutions were also found, while some substitutions weren’t taken into account when designing primers. This resulted in the Ff1/Fr5 primer pairing with high specificity for the target C. mintans xylanase sequence, without any high ambiguity.
“PCR Amplification and Cloning of the Genomic C. Minitans Xylanase Like Fragment, Ffaml
“PCR reactions were visualized under ultraviolet light and resolved using a 1.5% Agarose gel. Semi-purification was achieved by passing through a 1ml SEPHADEX DNA grade M (Pharmacia product no. 170045-01) column, and then ligated overnight into a pGEMT PCR vector system (Promega Corporation at 2800 Wood Hollow Road Madison, WI, 53711-5399 USA, product number A3600). The ligation mix was transformed into MAX EFFICIENCY DH5?COMPETENT CELLS (Life Technologies, product #18258-012) and recombinant vectors were screened by blue-white/ampicillin selection (white LacZ- cells are indicative of recombinant clones, transformants are selected through ampicillin resistance). The recombinant clones’ plasmid DNA (pDNA), was extracted using the WIZARD PLUS minipreppurification system (Promega Corporation product #18258-012) and sequenced using the universal 24-base M13 forward (5? cgc ggt ccc agt gac 3?) and the 24-base reverse (5? agc gga taa tt cac aca 3) sequencing primers with a fluorescent dye-terminator reaction kit, PE Applied Biosystems, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster Centre Drive, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster Center Drive, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, Foster City, USA, Applied Biosystems (ABI-TERMINATOR SEQUENCING READYE 93, California, USA, 944, USA, Applied Biosystems (850 Lincoln Centre Drive), Foster City, Foster City, CA, 94404 USA, Applied Biosystems (944)
“Characterizing C. Minitans Xylanaselike Genomic Fragment Sequence.”
“The sequence of C. minitans 693 bp genome fragment was extracted from the sequenced pGEM?T clone. It was then entered into an alignment search algorithm, BLAST, Altschul et al. 1997) to find homologous sequences. C. minitans’ genomic fragment was PCR-tested and showed high homology with F-family fungal xylanases originating in A. aculearus (likura et. al. 1998; MacCabe et. al. 1996; RuizRoldan et. al. 1998; Sheppard et. al. 1994).
“EXAMPLE 2”
“Assessing the Presence, Size, and Abundance of a C. Minitans Xylanase Hybridizing Transcript
“RNA isolation from C. Minitans strains A11-3B 2A2, 2134, and A10-4”
“Northern Hybridization Analysis for Total C. Minitans’ RNA”
“Ten micrograms of total RNA was loaded and resolved on a 1.5% agarose gel containing 1.9% formaldehyde, and 0.02M MOPS (3-(N-morpholino)propane sulfonic acid) buffer. The RNA was transferred onto a nylon membrane (MAXIMUM STRENGTHNYTRAN, Schleicher & Schuell PO Box 2012 Keene N.H. 03431, USA product #77404) using Northern transfer (Sambrook et. al. 1989). It was then immobilized by UV crosslinking.
The resulting autoradiogram showed significant expression of a 1.3 kb transcription which hybridized with the C. mintans genomic xylanase fragment. This indicates that C. minitans strain C11-3B 2A2 had abundant expression of a xylanase like transcript in the presence ground sclerotia (S. sclerotiorum) as the sole source of carbohydrate. The xylanase like transcript was also found in total RNA from wild-type strain 2134, but it was less prevalent in strain A10-4 grown in minimal Czepek?Dox medium with ground sclerotia S. sclerotiorum. C. minitans strains 2134 (wild type) M11-3B2A2 were not able to produce a xylanase like transcript.
“EXAMPLE 3”
“Constructing and Screening a C. mintans cDNA library in the Isolation and Sequencing the Full-length Xylanase Transcript Sequencing (cxy1)”
“C. minitans strain 2A2 was grown in Czepek-Dox broth media that contained 1% ground Sclerotia of S.clerotiorum. It was kept for 15 days. As in Example 2, mycelia was taken and ground in liquid nitrogen. TRIZOL solution (Life Technologies) was used to extract total RNA. The total RNA mixture was then purified using a cellulose bound oligo-dT purification device (MESSAGEMAKER, Life Technologies product #10551-018). First strand cDNA was synthesized from the purified mRNA using SUPERSCRIPT II RNAse H reverse-transcriptase (Life Technologies, product #18053-017) and was cloned unidirectionally into a ?ZAP-cDNA GIGAPACK III GOLD cloning kit (Stratagene product #200450). Bacteriophage clones are packaged with?phage GIGAPACKIII GOLD packaging extract (Stratagene product #200450). They were then titered onto NZY media (1% (w/v), NZ amine 0.5% (w/v), NaCl 0.5% (w/v), 0.5% (w/v), yeast extract (Bacto), 0.2%(w/v), MgSO4.7H2 0. pH 7.0). On 15 cm Petri plates, phages were plated at a density 50,000 per plate.
“EXAMPLE 4”
“Characterization of cxy1 DNA by Amino Acid Sequence Alignment: Characterization and Structural Analysis”
“Phylogenetically, fungal species can be quite different in terms of their non-functional genomic sequences. However, functional sequences that are required for specific biological functions (such as catalytic or functional domains of cellulolytic enzymes) are often high in conservation. Comparing amino-acid sequences with other fungal xylanases may help identify functional domains within Cxy1.
Cxy1 seems to have a similar structural layout to many F-family fungal Fxylanases. Typical fungal xylanases, such as the Aspergillus kawachii xylanase XynA, (Ito el., 1992), and the Emericella nidulans xylanase XylnC, (MacCabe et., 1996), have a unique Nterminus that is followed by a catalytic domain at the C-terminus. Cxy1 seems to have extensive homology in the C-terminus catalytic area. However, Cxy1’s xylanase may also include a carbohydrate binding domain (CBD). This domain is located between amino acids 22 and 52. It is flanked by WSQCGG and WYFQC (SEQID NO: 2, amino acid 22-27) and WYFQC motifs. These sequences are very similar to those of microbial glycanases (Gilkes et. al. 1991). The conservation of the entire sequence suggests that it may still have carbohydrate binding function. The CBD domain is used in cellulolytic and hemicellulolytic enzymes to tether enzyme to a substrate. This enhances substrate specificity, as well as degradative activity. However, the presence of a CBD-like sequence does not necessarily mean that an enzyme will exhibit cellulolytic activities. For instance, the Pseudomonas fluorescens xylanase Xyna contains a cellulose-binding domain, but does not demonstrate hydrolytic activity towards carboxymethylcellulose (CMC)(Gilbert et al., 1988; Hall et al., 1989). Some xylanases have dual cellulolytic/xylanolytic functions. Clostridium thermocellurn F family xylanase, XynZ, exhibits xylanase activation with high specificity but low cellulase activity (Greprinet and al., 1988).
“The Cxy1 CBD has been linked to the Cterminus catalyticdomain by a unique linker sequence (SEQ ID No:2, amino acids 53 through 83), which may play a role in coordination of the tertiary CBD and catalytic domain during the xylan catalysis process (FIGS. 2A, 2B). This structural arrangement is very similar to the F-family xylanase homologues Ffam1 & Xyl3 found within the fungal phytopathogen Fursarium oxysporum. (Sheppard et. al. 1994; Ruiz?Roldan et. al. 1998). Cxy1 shows high homology with all F-family xylanases. The catalytic Cterminal domain (FIGS) is the most homological. 3A, 3B). Sequence conservation does not only apply to fungal F family cellulases but also extends to prokaryotic F.family xylanases like the thermostable xylanase XylA, which is found in the eubacteria Thermomonospora Alba (Blanco et. al., 1997) and the C. thermocellum XynY, (Fontes et. al., 1995).
“EXAMPLE 5”
“Assaying Cxy1 activity in Escherichiacoli”
Many eukaryotic enzymes need extensive post-translational modifications to maintain their functional properties. Functional reconstitution of eukaryotic protein in prokaryotic systems is often blocked by post-translational processing mechanisms like glycosylation (additions of glycosyl group to thr/ser(O-linked), or asn(N-linked). residues)) and proteolytic destruction. A cxy1-cDNA-llacZ fusion produced a significant amount of functional xylanase in Escherichia Coli. This suggests that Cxy1 activity is not dependent on post-translational modifications. It also indicates that Cxy1 can be used in conjunction with prokaryotic expression systems.
“Sequence Verification for the lacZ/cxy1fusion Clone”
“Several pBLUESCRIPT-cxy1 clones were created by removing the phagemid from a lambda vector from the C. mintans M11-3B2A2 cDNA database. To determine if the cxy1 insert would transcribe into a translated Cxy1 product, we sequenced pBLUESCRIPT cxy1 cells.
“Assaying the Xylanolytic Activity for the Cxy1/LacZ Fusion protein from E. Coli [strain DH5?]”
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