Patent Application
20090325240
The composition and diversity of biomass for producing cellulosic ethanol requires multiple enzyme systems, in very high concentrations, to release constituent sugars from which cellulosic ethanol is made. All currently available enzymes for cellulosic ethanol are produced in expensive fermentation systems and are purified in a process very similar to biopharmaceuticals like insulin. Therefore, reagent grade enzymes for ethanol production are extremely expensive. For example, B-glucosidase, pectolyase and cellulase are currently sold by Novozyme through the Sigma catalog for $124,000, $412,000 and $40,490 per kg, respectively. These enzymes are sold as formulations to bio-refineries without disclosing the actual enzyme components. Therefore, the actual cost for each enzyme, sold in bulk quantities, is not publicly available. Most industrial estimates for enzymes to produce cellulosic ethanol are in the $2 to $3 per gallon range, making large scale use cost prohibitive. Current capacity of fermentation systems will also be a major limitation. With increase in demand for enzymes and limited production capacity, the enzyme cost is likely to increase further.
A major limitation for the conversion of this biomass to ethanol is the high cost and large quantities of enzymes required for hydrolysis. B-glucosidase, pectolyase and cellulase are currently sold by Novozyme or other industries through the Sigma catalog for $124,000, $412,000 and $40,490 per kg, respectively. Therefore, the US DOE has long identified the cost of enzymes and their high loading levels required for most lingo-cellulosic feedstocks as one of the major barriers to cellulosic ethanol production. Currently, all commercially-available enzymes are produced through a fermentation process. Unfortunately, the building and maintenance of the fermentation production process is very expensive, costing $500M-$900M in upfront investment. No viable alternative to fermentation technology has yet emerged for mass-producing critical, yet prohibitively expensive industrial enzymes. This void in the marketplace for an alternative process is addressed directly by this proposal.
E. coli vs chloroplast derived enzymes at different protein concentrations of crude extracts. (a) Enzyme kinetics of cpCelD and rCelD using carboxymethyl cellulose (2%) substrate. The reaction mixture contained increasing concentration of cpCelD and rCelD TSP (μg/ml) with 10 mM CaCl2 and 50 mM sodium acetate buffer, pH 6.0. Enzyme hydrolysis was carried out for 30 minutes at 60° C. Figure inset shows enzyme kinetics saturation point for cpCelD TSP amount (μg/ml) towards CMC (2%). Eppendorf tubes with reaction mixture shown in inset represents, 1 untransformed plant, 2 and 3 rCelD and cpCelD 10 μg TSP. (b) Effect of cpPelB, cpPelD, rPelB, and rPelD on hydrolysis of 5.0 mg/ml sodium polygalacturonate substrate. The reaction mixture contained increasing concentration of cpPelB, cpPelD, rPelB, and rPelD (μg/ml) in 20 mM Tris-HCl buffer (pH 8.0). Sodium polygalacturonate (Sigma) was measured using DNS method and measured from the D-galacturonic acid standard graph. Enzyme hydrolysis was carried out for 2 hour at 40° C. on rotary shaker at 150 rpm.
Certain embodiments of the invention address the major problems discussed above by producing all required enzymes in plants or bacteria or a combination of both, thereby dramatically alleviating the cost of fermentation and purification. According to one embodiment, the invention pertains to a method of degrading a plant biomass sample so as to release fermentable sugars therein. The method involves obtaining a plant degrading cocktail comprising at least two cell extracts, each cell extract having an active plant degrading compound that was recombinantly expressed in cells from which each said cell extract is derived. The at least two cell extracts are either plant extracts or bacterial extracts, or a combination of both. The plant degrading cocktail is admixed with the biomass sample to release fermentable sugars. In a more specific embodiment, the plant degrading cocktail includes cell extracts that include plant degrading enzymes such as cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, lipase, maltogenic alpha-amylase, pectolyase, or compounds that facilitate access of such enzymes, or so called, accessory plant degrading compounds, including but not limited to cutinase or expansin (e.g. swollenin). The inventors have found that such accessory plant degrading compounds serve to facilitate the action of plant degrading enzymes which synergistically elevates the amount of fermentable sugars produced for any given biomass sample.
Plant cell extracts will in most cases include an amount of ribulose-1,5-bisphosphate carboxylase_oxygenase (rubisco) from the plant cells from the plant cell extracts are derived. Rubisco is the most prevalent enzyme on this planet, accounting for 30-50% of total soluble protein in the chloroplast; it fixes carbon dioxide, but oxygenase activity severely limits photosynthesis and crop productivity (Ogren, W. L. (2003) Photosynth. Res. 76, 53-63, 2. Spreitzer, R. J. & Salvucci, M. E. (2002) Annu. Rev. Plant Biol. 53, 449-475). Rubisco consists of eight large subunits (LSUs) and eight small subunits (SSUs). The SSU is imported from the cytosol, and both subunits undergo several posttranslational modifications before assembly into functional holoenzyme (Houtz, R. L. & Portis, A. R. (2003) Arch. Biochem. Biophys. 414, 150-158.).
The use of genetic transformation of plants is generally not considered a viable alternative to the conventional fermentation processes for producing plant degrading enzymes in light of the fact that the expressed proteins would be deleterious to plant life and growth. The inventors have endeavored to devise a method of expressing plant degrading enzymes in such a way that does not disrupt the plant cell. It is the inventors' belief that the present invention is the first demonstration of viable plant degrading enzyme expression in plants. The inventors have realized that the expression of many plant degrading enzymes can be expressed in chloroplasts without adverse effects on the plant. The chloroplasts appear to insulate the plant cell from damage from the enzymes. Though expression in chloroplasts is exemplified herein, unless specifically stated, embodiments of the present invention should not be construed to be limited to chloroplast expression of plant degrading enzymes. Certain embodiments related to a combination of plant and bacterial extracts from cells engineered to recombinantly express plant degrading compound(s).
The term “recombinantly expressed” as used herein refers to production of a polypeptide from a polynucleotide that is heterologous to the cell in which the polynucleotide has been transfected. Recombinant expression may result from heterologous polynucleotides that are stably transformed in the genome of the cell, or genome of the cell organelle, or which are merely present in the cell via a transfection event.
The term chloroplast is interpreted broadly to cover all plastids, including proplastids, etioplasts, mature chloroplasts, and chromoplasts.
A comprehensive cellulase system consists of endoglucanases, cellobiohydrolases and beta glucosidases. The cellobiohydrolases and endoglucanases work synergistically to degrade the cellulose into cellobiose, which is then hydrolysed to glucose by the beta glucosidases. Cellobiase, or beta-glucosidase, activity is responsible for the formation of glucose from cellobiose and plays an important role in cellulose degradation by relieving the end product (cellobiose) inhibition. Gene sequences for most of these enzymes, from different microorganisms, have been deposited in public data bases.
Pectins, or pectic substances, are collective names for a mixture of heterogeneous, branched and highly hydrated polysaccharides present as one of the major components in plant cell walls. These polysaccharides comprise mostly neutral sugars, such as arabinan, galactan, andarabino galactan. Activepectolytic enzyme preparations have the following enzymes: Two alpha-L-rhamnohydrolases, polygalacturonase, pectin methylesterase, endo-pectate lyase (pectintranseliminase), pectin lyase and small percent of xylanase. Nucleotide sequence for pectin degrading enzymes, xylanases, cellulases are available in public data bases. See Example 9 herein for discussion on sequences.
The inventor has realized that enzyme requirements are very different for each type of biomass used in cellulosic ethanol production. Accordingly, certain embodiments of the present invention relate to cocktails of enzymes obtained from plant expression and/or bacteria expression that the inventors have developed to be particularly effective for the targeted biomass material.
Biomass sources that can be degraded for ethanol production in accordance with the teachings herein, include, but are not limited, to grains such as corn, wheat, rye, barley and the grain residues obtained therefrom (primarily leftover material such as stalks, leaves and husks), sugar beet, sugar cane, grasses such as switchgrass and Miscanthus, woods such as poplar trees, eucalyptus, willow, sweetgum and black locust, among others, and forestry wastes (such as chips and sawdust from lumber mills). Other biomasses may include, but are not limited to, fruits including citrus, and the waste residues therefrom, such as citrus peel.
Throughout this document, tobacco is referred to as an exemplary plant for expressing plant degrading enzymes. However, unless specifically stated, embodiments of the invention should not be construed to be limited to expression in tobacco. The teachings of gene expression taught herein can be applied to a wide variety of plants, including but not limited to tobacco; lettuce, spinach; sunflower; leguminous crops such as soybean, beans, peas, and alfalfa; tomato; potato; carrot; sugarbeet; cruciferous crops; fibre crops such as cotton; horticultural crops such as gerbera and chrysanthemum; oilseed rape; and linseed.
In one embodiment, genes from Aspergillusniger, Aspergillus aculeatus, Trichodermareesei andor Clostridium thermocellum encoding different classes of enzymes are isolated using gene specific primers. In order to express different classes of genes in a chloroplast of interest, the following strategies are used. The chloroplast vector contains flanking sequences from the chloroplast genome to facilitate homologous recombination. In one embodiment, foreign genes are integrated individually into the spacer region of chloroplast genome. The coding sequence of different enzymes can be regulated by appropriate regulatory sequences. Recombinant plasmids will be bombarded into tobacco to obtain transplastomic plants.
In a specific embodiment, powdered tobacco leaves are used as enzyme sources for commercial evaluation of ethanol production from a biomass source. In a more specific embodiment, the biomass source is a grain such as corn, a grass, or is citrus waste.
For chloroplasts that are transformed, it has been realized that obtaining homplasmy with respect to the transgenic chloroplasts is desired. The transgene integration and homoplasmy is confirmed by PCR and Southern blot analysis, respectively. Expression of the transgenes is confirmed by western blot analysis and quantified by ELISA. The protein extract from transplastomic tobacco plants is tested for its ability to degrade citrus waste biomass. Based on the results, more enzyme classes are added to increase the breakdown of plant biomass to sugars for fermentation to ethanol. Each tobacco plant, engineered to produce an enzyme, will be able produce a million seeds, to facilitate scale up to 100 acres, if needed. Homogenized plant material, such as powdered tobacco leaves or plant extracts, or purified enzymes from plant material are used as the enzyme source for commercial evaluation of ethanol production from citrus waste.
While current methods involve placing a foreign gene in the plant cell nucleus, CT transforms the genome of the approximately 100 chloroplasts that are within each tobacco plant cell. Each tobacco plant chloroplast contains about 100 copies of the chloroplast's genetic material, so the amount of protein (in this case, enzyme proteins used to break down biomass into sugar for ethanol production) is increased exponentially. This is the primary reason why massive volume production of cell wall degrading enzymes for cellulosic ethanol production is so cost effective. Secondly, tobacco has large volume biomass (40 metric tons of leaves per acre) and it can be harvested multiple times during a given growing season in Florida. And, as previously mentioned, it is easy to plant 100 acres from a single tobacco plant. Lastly, because chloroplasts are inherited maternal, they are not functional in the tobacco plant's pollen.
According to one embodiment, the invention pertains to a method of degrading a plant biomass sample to release fermentable sugars. The method includes obtaining a plant degrading cocktail having at least one chloroplast genome or genome segment having a heterologous gene that encodes cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase or pectolyase, or a combination thereof, and wherein said plant material has cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase or expansin, or a combination thereof, that has been expressed in a plant from which said plant material is derived; and admixing said plant degrading material with said biomass sample. In a more specific embodiment, the enzyme or combination of enzymes pertains to more than 0.1 percent of the total protein in the plant material.
According to another embodiment, the invention pertains to a method of producing a plant biomass degrading material sufficient to release fermentable sugars, the method including producing at least one plant comprising chloroplasts that express cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof; harvesting said plant; and processing said plant to produce an enzyme source suitable for mixing with and degrading a biomass sample. In a specific embodiment the plant is tobacco; lettuce, spinach; sunflower; fibre crops such as cotton; horticultural crops such as gerbera and chrysanthemum; leguminous crops such as soybean, beans, peas, and alfalfa; tomato; potato; sugarbeet; cruciferous crops, including oilseed rape; and linseed. In a more specific embodiment, plant is tobacco.
One aspect of the invention is to provide an abundant inexpensive source of enzyme for degrading biomass. Accordingly, the plant or bacterial material in which plant degrading compounds have been expressed may be processed by drying and powderizing the plant or a portion thereof. In another embodiment, crude liquid extracts are produced from the plant and/or bacterial material. In alternative embodiments, the plant degrading material may be enzymes that have been purified fully or partially from the plant and/or bacteria in which they are expressed. However, providing the plant degrading material as a dry form or as crude extract of the plant and/or bacterial material avoids the need for time-consuming and potentially expensive purification steps. In this way, the plant material has a longer shelf life and may easily be mixed with the plant biomass sample according to conventional plant degrading and fermenting processes.
In one specific embodiment, the method of producing a plant entails producing a first plant with chloroplasts transformed to express a first enzyme and a second plant with chloroplasts transformed to express a second enzyme. Plant material from both first and second plants may be combined to produce a plant degrading sample that includes more than one plant degrading enzyme. In a more specific embodiment, the invention pertains to a method of producing a plant that entails at least two of the following: producing a first plant comprising chloroplasts that express cellulase, producing a second plant comprising chloroplasts that express lignanse, producing a third plant comprising chloroplasts that express beta-glucosidase; producing a fourth plant comprising chloroplasts that express hemicellulase; producing a fifth plant comprising chloroplasts that express xylanase; producing a sixth plant comprising chloroplasts that express alpha-amylase; producing a seventh plant comprising chloroplasts that express amyloglucosidase; producing an eighth plant comprising chloroplasts that express pectate lyase; producing a ninth plant comprising chloroplasts that express cutinase; producing a tenth plant comprising chloroplasts that express lipase; producing an eleventh plant comprising chloroplasts that express maltogenic alpha amylase, producing a twelfth plant comprising chloroplasts that express pectolyase and/or a thirteenth plant comprising chloroplasts that express expansin (e.g. swollenin).
As alluded to above, the inventors have recognized that according to certain embodiments, plant derived enzymes are augmented with plant degrading enzymes recombinantly expressed in bacteria. Thus, a plant degrading cocktail may include enzymes recombinantly expressed in plants and enzymes that are recombinantly expressed in bacteria, such as but not limited to E. coli.
According to a further embodiment, the invention pertains to a plant material useful for degrading a plant biomass, the material including at least one chloroplast genome or genome segment having a heterologous gene that encodes cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof; and wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof. In a more specific embodiment, the plant material includes at least two of the following: a first chloroplast genome or genome segment having a heterologous gene that encodes cellulase, a second chloroplast genome or genome segment having a heterologous gene that encodes lignanse, a third chloroplast genome or genome segment having a heterologous gene that encodes beta-glucosidase; a fourth chloroplast genome or genome segment having a heterologous gene that encodes hemicellulase; a fifth chloroplast genome or genome segment having a heterologous gene that encodes xylanase; a sixth chloroplast genome or genome segment having a heterologous gene that encodes alpha-amylase; a seventh chloroplast genome or genome segment having a heterologous gene that encodes amyloglucosidase; an eighth chloroplast genome or genome segment having a heterologous gene that encodes pectate lyase; a ninth plant chloroplast genome or genome segment having a heterologous gene that encodes cutinase; a tenth chloroplast genome or genome segment having a heterologous gene that encodes lipase; an eleventh chloroplast genome or genome segment that encodes maltogenic alpha-amylase, a twelfth chloroplast genome or genome segment having a heterologous gene that encodes pectolyase and a thirteenth chloroplast genome or genome segment having a heterologouse gene that encodes expansin (e.g. swollenin).
According to another embodiment, the invention pertains to a plant having a plant cell having a more than natural amount of cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase pectolyase or expansin, or a combination thereof, and wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, pectolyase, or expansin, or a combination thereof, active enzyme therein. In a more specific embodiment, the enzyme or combination of enzymes represents is more than 0.1 percent of the total protein of the cell. In an even more specific embodiment the enzyme or combination of enzymes represent more than 1.0 percent of the total protein in the cell.
In a further embodiment, the invention pertains to a plant derived composition comprising cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase and/or swollenin, and an amount of rubisco. In a specific embodiment, the rubisco to enzyme ratio ranges from 99:1 to 1:99.
In an additional embodiment, the invention pertains to commercial cultivars that recombinantly express one or more plant degrading compounds. Commercial cultivars of tobacco, such as, but not limited to, LAMD and TN90, produce substantially more leaf material than experimental cultivars. However, commercial cultivars are more sensitive to introduction of foreign genes. Surprisingly, the inventor, through significant trial and error, has been able to successfully transfect cells and induce expression of plant degrading compounds in commercial cultivars.
For the integration of transgenes, transcriptionally active spacer region between the trnI and trnA genes was used (
Transplastomic tobacco plants were obtained as described previously28, 29. Southern blot analysis was performed to confirm site specific integration of the pLD-pelB, pLD-pelD and pLD-celD cassettes into the chloroplast genome and to determine homoplasmy. Digestion of total plant DNA with SmaI from untransformed and transplastomic lines generated a 4.014 kb fragment untransformed (UT) or 6.410 kb in pelB, 6.377 kb in pelD or 7.498 kb fragment in celD when hybridized with the [32P]-labeled trnI-trnA probe, confirming site specific integration of the transgenes into the spacer region between the trnI and trnA genes (
Immunoblots with antibodies raised against PelA showed that PelB and PelD are immunologically related to PelA30. Therefore, PelA antibody was used to detect the expression of PelB and PelD, although their affinity was variable in transplastomic lines. All transplastomic lines showed expression of PelB or PelD protein. Expression levels did not change significantly corresponding to their enzyme activity depending on the time of harvest even though the PelB and PelD are regulated by light (
The activity of the enzyme varied significantly depending on the developmental stages and time of leaf harvest. Maximum enzyme activity was observed in mature leaves of PelB, PelD and CelD, with reduced activity in older leaves (
CelD enzyme activity was calculated using DNS reagent31 according to the IUPAC protocol32. The specific activity of cpCelD using 2% CMC substrate was 493 units/mg total soluble protein (TSP) or 100 mg leaf tissue, in crude extracts prepared from mature leaves harvested at 10 PM. Using the glucose hexokinase assay, which is highly specific for glucose, the specific activity was 4.5 units/mg TSP and 6.28 units/mg TSP, when 5% avicel and sigmacell solution respectively was used as substrate (at pH 6.0, 60° C.). Commercial cellulase enzyme (Trichoderma reesei, EC 3.2.1.4, Sigma) gave 4.2 units/mg and 3.43 units/mg solid for the same substrate.
Both plant and E. coli extracts showed optimal activity at 2.5 mg/ml PGA (
The crude extract (4-5 μg TSP) from plant or E. coli was used to study the effect of pH and temperature on the activity of enzymes. The optimal temperature for the E. coli and chloroplast derived pectate lyase under the standard assay conditions was 40° C. and the optimal pH was 8.0. Plant derived pectate lyases showed a pH optimum of 6.0 in the presence of 1 mM CaCl2. The E. coli crude extracts showed an optimal pH of 8.0 irrespective of presence or absence of CaCl2 in the reaction (
CpCelD activity with 2% CMC was measured at different pH and temperature. The cpCelD showed pH optima between pH 5.0 to pH 7.0 (
E. coli crude extract containing CelD enzyme showed decrease in enzyme activity when the reaction mixture contained more than 10 μg TSP, where as plant crude extract containing CelD released more reducing sugar with increasing protein concentration (
Before evaluation of enzyme cocktails, activity of each enzyme was tested independently with an appropriate substrate. Chloroplast or E. coli expressed endoglucanase (cpCelD or rEg1) alone did not release any detectable glucose from filter paper but when mixed together up to 19% of total hydrolysis was observed (
The enzyme cocktail that released highest glucose equivalents with filter paper (except rEg1) was tested on processed wood substrate. After 24 hour hydrolysis, 31% of total hydrolysis was observed with this cocktail (
According to one embodiment, the invention pertains to a method for digesting a wood-based biomass sample comprising obtaining a plant material comprising endoxylanase or acetyl xylan esterase, or a combination thereof, that has been expressed in a plant from which all or a portion of said plant material is derived; and admixing said plant material with said wood based biomass sample. The method of this embodiment may further include admixing with the wood-based biomass sample, either prior to or contemporaneous to, admixture with the endoxylanase and/or acetyl xylan esterase plant material, a plant material comprising a pectate lyase that has been expressed in a plant from which all or a portion of said plant material is derived. The plant material may comprise rubisco.
Another embodiment pertains to a plant degrading enzyme cocktail useful in digesting a wood-based biomass sample comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.
The enzyme cocktail of endoglucanase (cpCelD), exoglucanase, swollenin and beta-glucosidase released up to 24% of total hydrolysis with citrus peel (
According to one embodiment, the invention pertains to a method for digesting a citrus biomass sample comprising obtaining a plant material comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase, or a combination thereof, that has been expressed in a plant from which all or a portion of said plant material is derived; and admixing said plant material with said citrus biomass sample.
Another embodiment pertains to a plant degrading enzyme cocktail useful in digesting a citrus biomass sample comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.
Isolation of Genes and Construction of Plastid Transformation Vectors
Genomic DNA of Clostridium thermocellum and Trichoderma reesei was obtained from ATCC and used as template for the amplification of different genes. Gene specific primers using a forward primer containing a NdeI site and a reverse primer containing a XbaI site for cloning in the pLD vector were designed for celD, celO and lipY genes. The mature region of cellulose genes celD (X04584) and celO (AJ275975) were amplified from genomic DNA of Clostridium thermocellum. LipY (NC—000962) was amplified from genomic DNA of Mycobacterium tuberculosis. Overlapping primers were designed for the amplification of various exons of egl1 (M15665), egI (AB003694), swoI (AJ245918), axe1 (Z69256), xyn2 (X69574) and bgl1 (U09580) from genomic DNA of Trichoderma reesei using a novel method. Full length cDNA of these genes was amplified from different exons by a novel PCR based method using the forward of first exon and reverse of last exon containing a NdeI site and XbaI site respectively. Pectate lyase genes pelA, pelB & pelD from Fusarium solani with similar restriction sites were amplified using gene specific primers from pHILD2A, pHILD2B30 and pHILD2D45 respectively. A similar strategy was used to amplify cutinase gene46 from recombinant clone of Fusarium solani. All the full length amplified products were ligated to pCR Blunt II Topo vector (Invitrogen) and were subjected to DNA sequencing (Genewiz). Each gene cloned in Topo vector was digested with NdeI/XbaI and inserted into the pLD vector17, 47 to make the tobacco chloroplast expression vector.
Regeneration of Transplastomic Plants and Evaluation of Transgene Integration by PCR and Southern Blot
Nicotiana tabacum var. Petite Havana was grown aseptically on hormone-free Murashige and Skoog (MS) agar medium containing 30 g/l sucrose. Sterile young leaves from plants at the 4-6 leaf stages were bombarded using gold particles coated with vector pLD-PelB, pLD-PelD and pLD-CelD and transplastomic plants were regenerated as described previously28, 29. Plant genomic DNA was isolated using Qiagen DNeasy plant mini kit from leaves. PCR analysis was performed to confirm transgene integration into the inverted repeat regions of the chloroplast genome using two sets of primers 3P/3M and 5P/2M, respectively17. The PCR reaction was performed as described previously17, 29. Leaf from the PCR positive shoots were again cut into small pieces and transferred on RMOP (regeneration medium of plants) medium containing 500 mg/l spectinomycin for another round of selection and subsequently moved to MSO (MS salts without vitamins and growth hormones) medium containing 500 mg/l spectinomycin for another round of selection to generate homoplasmic lines. Southern blot analysis was performed to confirm homoplasmy according to lab protocol48. In brief, total plant genomic DNA (1-2 μg) isolated from leaves was digested with SmaI and hybridized with 32P α[dCTP] labeled chloroplast flanking sequence probe (0.81 kb) containing the trnI-trnA genes. Hybridization was performed by using Stratagene QUICK-HYB hybridization solution and protocol.
Immunoblot Analysis
Approximately 100 mg of leaf was ground in liquid nitrogen and used for immunoblot analysis as described previously48. Protein concentration was determined by Bradford protein assay reagent kit (Bio-Rad). Equal amounts of total soluble protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. The transgenic protein expression was detected using polyclonal serum raised against PelA in rabbit.
E. coli Enzyme (Crude) Preparation
E. coli strain (XL-10 gold) harboring chloroplast expression vectors expressing rCelD, rEg1 (EC 3.2.1.4), rCelO (EC 3.2.1.91), rXyn2 (EC 3.2.1.8), rAxe1 (EC 3.1.1.72), rBgl1 (EC 3.2.1.21), rCutinase (EC 3.1.1.74), rLipY (lipase, EC 3.1.1.3), rPelA, rPelB, rPelD (EC 4.2.2.2) or rSwo1 was grown overnight at 37° C. Cells were harvested at 4° C. and sonicated four times with 30 s pulse in appropriate buffer (50 mM sodium acetate buffer with pH 5.5 for CelD, Eg1, CelO, Swo1, Xyn2, Axe1, Bgl1, 100 mM Tris-Cl with pH 7.0 for cutinase, lipase, PelA, PelB and PelD) containing protease inhibitor cocktail (Roche) and sodium azide (0.02%). Supernatant was collected after centrifugation at 16,000×g for 10 minutes and protein concentration was determined.
Enzyme Preparation from Tobacco Transplastomic Leaf Material
Fresh green leaves were collected and ground in liquid nitrogen. Total soluble protein was extracted in 50 mM sodium acetate buffer, pH 5.5 for cpCelD, cpXyn2 or 100 mM Tris-Cl buffer, pH 7.0 for PelD and PelB. All buffers contained protease inhibitor cocktail (Roche) and sodium azide (0.02%). Total soluble protein was filtered using 0.22 μm syringe filter, Protein concentration (mg/ml) in TSP was determined using Bradford method.
Enzyme Assays for Pectate Lyase B and Pectate Lyase D
Pectate lyases B and D were assayed spectrophotometrically by measuring the increase in A23530, 49, 50. Kinetics of the pectate lyase B and D were studied to optimize substrate concentration (0.0-2.5 mg) under identical protein and cofactor concentration. The reaction mixtures contained 1 ml of 50 mM Tris-HCl buffer (pH 8.0) with 1 mM CaCl2 (freshly prepared), 1 ml of 0.0-2.5 mg/ml sodium polygalacturonate (Sigma) and 0.5 ml of suitably diluted enzyme solution. Measurements were carried out at 40° C. One unit of enzyme was defined as the amount of enzyme which forms 1 μmol of product per min with a molar extinction coefficient of 4,600 μmol−1 cm−1. Kinetic studies were carried out in 50 mM Tris-HCl buffer, pH 8.0 at 40° C. Kinetic parameters (Km & Vmax) were calculated using non linear regression using Graphpad Prism 5.0. The initial slopes of each substrate concentration were calculated, where as the velocity (units/mg/min) was defined through the release of unsaturated galacturonic acid. The temperature optimization for pectate lyase B and D activity was carried out in 50 mM Tris-HCl buffer, pH 8.0 at different temperatures ranging from 30° C. to 70° C. In each case, the substrate was pre-incubated at the desired temperature for 5 min. In order to study the thermal stability of the enzyme, buffered enzyme samples were incubated for fixed time period at different temperatures.
The pH optimum of the pectate lyase B and D was measured at 40° C. using different buffers ranging from pH 6 to 10, with the same ionic strength. The stability of the crude extract of the enzyme was optimized by incubating the enzyme at the different pH. The influence of the cofactor CaCl2 on pectate lyase activity was studied by conducting the reactions in its presence and absence at different pH and temperature.
Enzyme Assay for CelD and Commercial Cocktail (Celluclast 1.5 L and Novozyme 188)
Cellulase enzyme activity of cpCelD was determined by incubating crude extract in 2% carboxylmethylcellulose, avicel and sigmacell (Sigma) as substrate according to IUPAC recommendations32 in 50 mM sodium acetate buffer pH 6.0 and incubated at 60° C. for 30 minutes for CMC and 2 hours for avicel and sigmacell. Enzyme units of commercial cocktails Celluclast 1.5 L and Novozyme 188 were determined using 2% CMC, under identical assay conditions. Reducing sugar amount was determined using 3,5-dinitrosalicylic acid31. D-glucose and D-galacturonic acid were used as standard to measure release of glucose equivalents and unsaturated galacturonic acid molecules. CMC (2%) was used in determining the pH and temperature activity profile of cpCelD. One unit of enzyme was defined as the amount of enzyme that released 1 μmole glucose equivalents per minute/ml. Cellulase unit calculation for avicel and sigmacell was based on glucose hexokinase method according to the manufacturer's protocol (Sigma).
Enzymatic Hydrolysis of Filter Paper, Processed Wood and Citrus Peel
Enzyme assays were carried out either with one enzyme component or as cocktail on filter paper, processed wood and orange peel and released reducing sugar was determined using DNS method. Orange peel prepared from Valencia orange (Citrus sinensis cv Valencia) fruit was air dried overnight and ground in liquid nitrogen. Ground Valencia orange peel and pretreated wood biomass were washed several times in distilled water until no reducing sugar was detected by DNS reagent as well as by glucose hexokinase method.
For enzymatic digestion, 50-200 mg of processed wood sample or ground orange peel was used. Crude extracts containing enzymes from E. coli and plants were used in the cocktail for hydrolysis. End product reducing sugar was determined using DNS reagent31 and D-glucose as standard. Ampicillin and kanamycin 100 μg/ml was added to prevent any microbial growth during the long durations of enzyme hydrolysis. Commercial enzyme cocktails Celluclast 1.5 L and Novozyme 188 were tested for hydrolysis of citrus peel and processed wood in the same assay conditions used for enzyme cocktails from crude extracts. Enzyme units of Celluclast 1.5 L and Novozyme 188 used for hydrolysis assays were equivalent to cpCelD enzyme units (based on CMC hydrolysis) present in cocktails of crude extracts. In all experiments control assays contained substrate without enzyme or enzyme without substrate. All experiments and assays were carried out in triplicate.
The inventors have used coding sequences from bacterial or fungal genomes to create chloroplast vectors. A novel PCR based method was used to clone ORFs without introns from fungal genomic DNA. E. coli expression system was used to evaluate functionality of each enzyme independently or their efficacy in enzyme cocktails before creating transgenic lines; enzymes of fungal origin were active without any need for post-translational modifications (disulfide bonds or glycosylation). The phenotypes of homoplasmic transplastomic lines were normal and produced flowers & seeds. Based on three cuttings of tobacco in one year, 49, 64 and 10,751 million units of pectate lyase and endoglucanase activity can be obtained each year in an experimental cultivar. This yield could be increased 18-fold when these enzymes are produced in commercial cultivars. Based on USDA Economic Research Service, the cost of production of Burley tobacco in 2004 was $3,981 per acre. Because most enzymes for hydrolysis of plant biomass are active at higher temperatures, it is feasible to harvest leaves and sun dry them, as reported previously for chloroplast derived xylanase25. Based on enzyme activity observed in plant crude extracts in this study, there is no need for purification. Therefore, excluding processing cost, enzymes could be produced as low as 0.008 cents for PelB, 0.006 cents for PelD per enzyme unit (as defined in the commercial source Megazyme). This is 925-1,233 fold less expensive for pectate lyase B & D, when compared with current commercial cost (Megazyme produced from C. japonicus). While this cost or yield comparison may not be the same for all chloroplast-derived enzymes, this concept provides a promising new platform for inexpensive enzyme cocktails to produce fermentable sugars from lignocellulosic biomass.
To the best of our knowledge, this is the first study using enzyme cocktails expressed in plants for hydrolysis of lignocellulosic biomass to produce fermentable sugars and direct comparison of enzyme properties produced via fermentation or in planta, using identical genes and regulatory sequences. Majority of enzyme hydrolysis studies on natural substrates like pretreated wood, corn stover or wheat straw have used commercially available enzymes3, 42 or purified recombinant enzymes spiked with purified commercial enzymes40, 43. Accurate comparison of crude extract enzyme cocktails with commercial cocktails is not possible because of their unknown enzyme compositions. Therefore, equivalent enzyme units based on CMC hydrolysis was used as a basis for general comparison. ACCELLERASE™ 1000 (Genencor) was not available for this study because of required institutional agreements. Novozyme 188 purified enzyme cocktail did not yield any detectable glucose equivalents from processed wood and 11% more glucose equivalents with citrus peel, whereas Celluclast 1.5 L yielded 10% and 137% more with both biomass substrates than the crude extract cocktail, with equivalent enzyme units based on CMC hydrolysis. It is not surprising that crude extract cocktails performed equal to or better than purified enzyme cocktails because the later are produced by submerged fermentation from selected fungal strains that secerete several enzymes, simultaneously. According to Novozymes, a careful design of a combination of single component enzymes is necessary for rational utilization of these enzyme cocktails44.
The teachings set forth in Examples 1-8 may be adapted for preparing expression cassettes of the heterologous gene, constructing transformation vectors; transforming chloroplasts and chloroplast expression any of a numerous list of enzymes that the inventors have identified will be helpful in degrading plant biomass sources. Also, Applicants refer to U.S. Patent Pubs 20070124830 and 20060117412 for techniques of chloroplast transformation and expression of proteins.
A non-limiting list of exemplary enzymes includes the following in Table I:
In addition to the above list, attached tables II-VII set forth a list of different enzymes that may be used in conjunction with embodiments of the invention. The EC nos. are recognized designations (http://www.chem.qmul.ac.uk/iubmb/enzyme/ and NC-IUBMB) defining each of the enzymes with cross references to relevant polypeptide sequences and encoding polynucleotide sequences. Accession Nos. pertain to identifications sequences in either Genbank (one letter followed by five digits, e.g. M12345) or the RefSeq format (two letters followed by an underscore and six digits, e.g., NT—123456). Each of the sequences and related accession nos. are stored in the Corenucleotide division of the GenBank database system. An accession no. listed on table II-VII for a specific gene can be easily found by inputting the accession number in the search field of the Entrez system found at (http://www.ncbi.nlm.nih.gov/sites/gquery). In a specific embodiment, the sequences of the accession nos. specifically listed in tables II-VI include those in their original state or as revised/updated in the Genbank system as of Feb. 28, 2008. In other embodiments, it is contemplated that sequences may be revised/updated after Feb. 28, 2008 but otherwise recognized by the art as the more accurate sequence of the accession no. compared to the sequence stored prior to Feb. 28, 2008. In these other embodiments, sequences as revised but recognized as the true sequence and having at least a 95% sequence identity to the sequence as stored in database prior to Feb. 28, 2008 shall be considered as the sequence for the relevant accession no.
Applicants also incorporate by reference the ASCII text file entitled 10669-034 seqid filed with the present application. This text file contains sequence information of the accessions nos listed in Tables II-VII.
In addition, nucleotides and peptides having substantial identity to the nucleotide and amino acid sequences relating plant degrading enzymes (such as those provided in tables II-VII) used in conjunction with present invention can also be employed in preferred embodiments. Here “substantial identity” means that two sequences, when optimally aligned such as by the programs GAP or BESTFIT (peptides) using default gap weights, or as measured by computer algorithms BLASTX or BLASTP, share at least 50%, preferably 75%, and most preferably 95% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Non-limiting examples include glutamine for asparagine or glutamic acid for aspartic acid.
The term “variant” as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantial identity with the nucleotide or amino acid sequence of Attachment B, preferably 75% sequence identity and most preferably 90-95% sequence identity to the sequences of the present invention: provided said variant has a biological activity as defined herein. The variant may be arrived at by modification of the native nucleotide or amino acid sequence by such modifications as insertion, substitution or deletion of one or more nucleotides or amino acids or it may be a naturally occurring variant. The term “variant” also includes homologous sequences which hybridise to the sequences of the invention under standard or preferably stringent hybridisation conditions familiar to those skilled in the art. Examples of the in situ hybridisation procedure typically used are described in (Tisdall et al., 1999); (Juengel et al., 2000). Where such a variant is desired, the nucleotide sequence of the native DNA is altered appropriately. This alteration can be made through elective synthesis of the DNA or by modification of the native DNA by, for example, site-specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed, using techniques standard in the art.
In specific embodiments, a variant of a polypeptide is one having at least about 80% amino acid sequence identity with the amino acid sequence of a native sequence full length sequence of the plant degrading enzymes provided on the attached 10669-034SEDID ASCII file. Such variant polypeptides include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains, of the full-length amino acid sequence. Fragments of the peptides are also contemplated. Ordinarily, a variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with a polypeptide encoded by a nucleic acid molecule shown in Attachment B or a specified fragment thereof. Ordinarily, variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, or more.
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired identity between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by those that: (1) employ low ionic strength and high temperature for washing, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 degrees C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 degrees C., with washes at 42 degrees C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55 degrees C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55 degrees C.
“Moderately stringent conditions” are identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50 degrees C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated Tm of the hybrid under study. The Tm of a hybrid between an polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
T
m=81.50° C.−16.6(log10 [Na+])+0.41(% G+C)−0.63(% formamide)−600/l),
where l=the length of the hybrid in basepairs.
In a specific embodiment, stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C.
Having demonstrated by several examples that plant-degrading enzymes can be expressed in plants and bacteria, cocktails of enzymes can be produced that are especially adapted for degrading a targeted class of biomass. The inventors have discovered that certain combinations of selected enzymes are capable of hydrolyzing biomass materials in a synergistic fashion. For example, it has been discovered that two or more enzymes from a particular class work synergistically to degrade targeted material in biomass to increase the yield of fermentable sugars achieved. Based on the known composition of a target biomass sample, more than one enzyme in a particular class is included in the plant degrading cocktail. Furthermore, the amount of enzyme of a specific enzyme class known to degrade a specific compound in a biomass sample is increased relative to other enzyme in the cocktail as a function of the ratio of the specific compound/mass of biomass sample. General information concerning exemplary classes of enzymes are discussed below in relationship to the type of substrates on which they act. Equipped with the teachings and techniques discussed herein, one skilled in the art is able to determine cocktails suitable for a given biomass, and methods by which synergy of the enzymes in the cocktails can be determined.
Enzyme Assays
Most enzyme assays were determined spectrophotometrically by measuring the increase in A23530, 47, 48. Enzyme kinetics were studied to optimize substrate concentration (0-2.5 mg) under identical protein and cofactor concentrations. The reaction mixtures contained appropriate buffers, substrates and standard enzymes when commercially available. Measurements are made at different temperatures and pH. One unit of enzyme is defined as the amount of enzyme which forms 1 μmol of product per min with appropriate molar extinction coefficient. Kinetic parameters (Km & Vmax) are calculated using non linear regression using Graphpad Prism 5.0. The initial slopes of each substrate concentration are calculated where as the velocity (units/mg/min) is defined through the release of appropriate product. The temperature optimization is determined in proper buffers with required co-factors, substrates at optimal pH. In order to study the thermal stability of each enzyme, buffered enzyme samples are incubated for fixed time periods at different temperatures. Similarly, the pH optimum of each enzyme are measured at optimal temperatures using different buffers ranging from pH 6 to 10, with the same ionic strength. The stability of the crude extract of the enzyme is optimized by incubating the enzyme at the different pH. The influence of various cofactors for optimal enzyme activity is studied by conducting the reactions in the presence or absence of each cofactor, at different pH and temperature.
Endoglucanase, Cellobiohydralase and Beta-Glucosidase:
Cellulose is the most abundant renewable bioresource produced in the biosphere through photosynthetic process (˜100 billion dry tons/year)49-51. Cellulose biodegradation by cellulases and cellulosomes, produced by numerous microorganisms, represents a major carbon flow from fixed carbon sinks to atmospheric CO2 and studies have shown that the use of biobased products and bioenergy can achieve zero net carbon dioxide emission52, 53% Cellulose is a linear condensation polymer consisting of D-anhydroglucopyranose joined together by β-1,4-glycosidic bonds with a degree of polymerization (DP) from 100 to 20,000. Approximately 30 individual cellulose molecules are assembled into larger units known as elementary fibrils (protofibrils), which are packed into larger units called microfibrils, and these are in turn assembled into large cellulose fibers54, 55. The breakdown of biomass involves the release of long-chain polysaccharides, specifically cellulose and hemicellulose, and the subsequent hydrolysis of these polysaccharides into their component 5- and 6-carbon chain sugars15. One of the most important and difficult technological challenges is to overcome the recalcitrance of natural lignocellulosic materials, which must be enzymatically hydrolyzed to produce fermentable sugars2,7,8,56,57.
The mechanism of cellulose degradation involves three enzyme classes of cellulase. These include endoglucanases or 1,4-β-D-glucan-4-glucanohydrolases (EC 3.2.1.4) exoglucanase or 1,4-β-D-glucan cellobiohydrolases (E.C. 3.2.1.91), and β-glucosidase or β-glucoside glucohydrolases (E.C. 3.2.1.21)55,58,59. The combined actions of endoglucanases and exoglucanases modify the crystalline nature of cellulose surface over time, resulting in rapid changes in hydrolysis rates.60 The inventor has realized that the simultaneous action of these enzyme class on cellulose substrate completely breaks down the intramolecular β-1,4-glucosidic bonds of cellulose chains. This results in release of large amount of fermentable glucose molecules.
Endoglucanases (e.g CelD, Eg1): Endoglucanases cut at random at internal amorphous sites in the cellulose polysaccharide chain, generating oligosaccharides of various lengths and consequently expose new chain ends. Exoglucanases (e.g. CelO, Cbh2): Exoglucanases or cellobiohydrolases acts processively on the reducing or nonreducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Cellobiohydralases can also act on amorphous and microcrystalline cellulose structure having exposed cellulose chain ends. β-Glucosidases (e.g., BglA, Bgl1): It hydrolyze soluble cellobiose as well as longer cellodextrins molecules to glucose.
CelD, Egl1, EG1 (endo-glucanases) and CelO (cellobiohydrolase): Substrate: Carboxylmethylcellulose (2%) beta D-glucan (10 mg/ml), microcrystalline cellulose, Avicel, Sigma cellulose (all 50 mg/ml). At least two dilutions are made for each enzyme sample investigated in 50 mM sodium acetate buffer containing 10 mM CaCl2 and 20 μg BSA. In the assay, one dilution should release slightly more and one slightly less than 0.5 mg (absolute amount) of glucose. The sample is incubated at appropriate temperature for 30 minutes after adding 0.5 ml of substrate solution. After incubation, 0.5 ml of DNS is added to 0.5 ml of the reaction mixture followed by boiling for exactly 5 minutes in a boiling water bath along with enzyme blanks and glucose standards. Following boiling, 166 μl of 40% Rochelle salt is added and transferred immediately to a cold water bath not more than 30 minutes. The tube is mixed by inverting several times so that the solution separates from the bottom of the tube at each inversion. The mixture is measured at 575 nm in a spectrometer. The absorbance is translated (corrected if necessary by subtracting of the blank) into glucose production during the reaction using a glucose standard curve graph. Enzyme unit is defined as the amount of enzyme required to liberate 1.0 μmol per minute.32
Bgl1 (Trichoderma reesei): Beta-Glucosidase Assays:
At least two dilutions are made of each enzyme sample investigated. One dilution should release slightly more and one slightly less than 1.0 mg (absolute amount) of glucose in the reaction conditions. Add 1.0 ml of enzyme dilution in sodium acetate buffer pH 4.8 to 1.0 ml of cellobiose substrate. Incubate at 50° C. for 30-120 minutes. Stop assay by boiling in water bath for 5 minutes. Determine liberated glucose using glucose hexokinase (Sigma) method. One unit of enzyme is defined as 1.0 μmol of glucose released per minute from cellobiose. Substrate: 2-15 mM para-nitrophenyl D-glucoside in sodium acetate buffer pH 4.8. Add 0.3 ml of diluted enzyme solution to 0.6 ml 50 mM sodium acetate buffer and 0.3 ml para-nitrophenyl D-glucoside. Carry out the reaction at 50° C. for 10 minutes. Stop the reaction with 1M Na2CO3. Spectrophometrically measure the liberated p-nitrophenol at 410 nm using p-nitrophenol as standard. Construct the calibration curve for p-nitrophenol in the concentration range of 0.02-0.1 mM. Enzyme unit is defined as the amount of enzyme required to liberate 1.0 μmol of p-nitrophenol per min from pNPG.
Xylanase
Endo-xylanses: Xylan is one of the major components of the hemicellulose fraction of plant cell walls and accounts for 20-30% of their total dry mass. Unlike cellulose, xylan is a complex polymer consisting of a β-1,4-linked xylose monomers substituted with side chains. Hydrolysis of the xylan backbone is catalyzed by endob-1,4-xylanases (EC 3.2.1.8) and β-D-xylosidases (EC 3.2.1.37).6l Endoxylanases are capable of hydrolyzing the internal 1,4-β-bonds of the xylan backbone and thereby produce several xylo-oligomers of varying length. Complete hydrolysis of xylan involves endo-β-1,4-xylanase, β-xylosidase, and several accessory enzymes, such as a-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase and ferulic acid esterase.62, 63 For the biofuel industry, the inventor has realized that xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). Furthermore, hydrolysis of xylan molecules is very important step in the enzymatic hydrolysis hemicellulose and lignocellulosic materials because this gives larger accessibility for cellulases to act on exposed cellulose.
Xyn2 (Trichoderma reesei)
Substrate: Oat spelt xylan (1%); Birch wood xylan (1%, boil for 5 minutes until dissolved) D-xylose (0.01 to 1 mg/ml xylose) standard graph will be prepared by using DNS method. Assay: The enzyme sample is diluted in 1% xylan suspension with a total volume reaction up to 2 ml. Then the mixture is incubated for 30-120 minutes at 50° C. After incubation, 2 ml of DNS reagent is added to the reaction mixture followed by boiling for 5 minutes. The release of xylose concentration is measured spectrophotometrically at 540 nm. The enzyme unit is calculated by using standard formula64.
Cutinase:
Cutinase from the phytopathogenic fungus Fusarium solani pisi is an example of a small carboxylic ester hydrolase that bridges functional properties between lipases and esterases. Cutin, a polyester composed of hydroxy and hydroxy epoxy fatty acids containing 16 and 18 carbon atoms, is the major structural component of the protective barrier covering the surface of the aerial parts of plants65, 66. Cutinases not only degrade cutin polymers but also a large variety of short and long chain triacylglycerols are rapidly hydrolyzed. The enzyme belongs to the family of serine hydrolases containing the so called α/β hydrolase fold.67, 68 Hydrolyses of cutin, lipase and triacylglycerols molecules that present in large amount in citrus peel waste gives tremendous accessibility for enzymes like pectinases, cellulases and xylanases. Therefore cutinase is important enzyme component in the enzyme hydrolyses of citrus peel for biofuel production.
Cutinase assay67, 69: Substrate: p-nitrophenyl butyrate and p-nitrophenyl palmitate (0.01% or 10 mM). The enzyme is extracted from transplastomic plants in 100 mM Tris-HCl buffer (pH7.0) and 0.03% Triton X-100 will be added at the time of initiating enzyme assay. Reactions are performed by incubating for 10-15 min at 30° C. in a tube containing 1 ml of substrate (100 mM Tris-HCl, pH 7, 0.03% Triton X-100, and 0.01% p-nitrophenol butyrate and various amount of enzyme sample (ice cold). Release of p-nitrophenol is measured at 405 nm using p-nitrophenol as standard. Background activity is subtracted from the absorption reading if necessary. One unit of enzyme activity was defined as the amount that degrades 1 μmol of substrate per minute under standard conditions.
Mannanase:
Mannanase is used in the paper and pulp industry, for the enzymatic bleaching of softwood pulp, in the detergent industry as a stain removal booster, in the coffee industry for hydrolysis of coffee mannan to reduce the viscosity of coffee extracts, in oil drilling industry to enhance the flow of oil or gas, in oil extraction from coconut meat, in the textile industry for processing cellulosic fibers and in the poultry industry to improve the nutritional value of poultry feeds.70
Cell-wall polysaccharides can be converted into fermentable sugars through enzymatic hydrolysis using enzymes such as cellulases and hemicellulases and the fermentable sugars thus obtained can be used to produce lignocellulosic ethanol. Mannanase is a hemicellulase. Hemicellulose is a complex group of heterogeneous polymers and represents one of the major sources of renewable organic matter. Mannans are one of the major constituent groups of hemicellulose and are widely distributed in hardwoods and softwoods, seeds of leguminous plants and in beans. Hemicelluloses make up 25-30% of total wood dry weight. Hemicelluloses in softwoods are mainly galactoglucomannan, containing mannose/glucose/galactose residues in a ratio of 3:1:1 and glucomannan with mannose/glucose residues in the ratio of 3:171. Mannanases are endohydrolases that cleave randomly within the 1,4-β-D mannan main chain of galactomannan, glucomannan, galactoglucomannan, and mannan. Mannanases hydrolyzes the β-D-1,4 mannopyranoside linkages in β-1, 4 mannans. The main products obtained during the hydrolysis of mannans by β-mannanases are mannobiose and mannotriose. Additional enzymes, such as β-glucosidases and α-galactosidases are required to remove side chain sugars that are attached at various points on mannans. A vast variety of bacteria, actinomycetes, yeasts and fungi are known to produce mannanase. Among bacteria, mostly gram-positive, including various Bacillus species and Clostridia species and few strains of gram negative bacteria, viz. Vibrio and Bacteroides have also been reported. The most prominent mannan degrading group among fungi belongs to genera Aspergillus, Agaricus, Trichoderma and Sclerotium.70
The assay procedure for determination of the activity of Mannanase involves the incubation of the crude enzyme extract with the substrate (Galactomannan from Locust bean gum as a substrate). After the enzyme reaction the reducing sugars liberated are quantified and the enzyme activity is measured. Locust bean gum (0.5%) will be dissolved in citrate buffer (pH 5.3) and heated until boiled; this mixture is used as the substrate. The crude enzyme extract is incubated with the substrate at 50° C. for 5 minutes. The reducing sugars liberated in the enzyme reaction is assayed by adding Dinitro salicylic acid-reagent boiling for 5 min, cooling and measuring the absorbance at 540 nm72.
Another assay method involves carob galactomannan (0.2%) as substrate in sodium acetate buffer (pH 5). Crude enzyme extract is incubated with the substrate at 40° C. for 10 minutes. The reducing sugars liberated are measured by Nelson-Somogyi method and the enzyme activity is quantified73. In gel diffusion assay, gel plates are prepared by dissolving 0.05% (w/v) locust bean gum in citrate phosphate buffer (pH 5.0) along with Phytagar. Crude enzyme extract is transferred to the gel plates and incubated for 24 hours. Gels will be stained by using Congo red. Cleared zones (halos) on the plates indicated endo-beta-mannanase activity74.
Arabinofuranosidase 1 (ABF1)
ABF1 has been shown to have numerous potential uses. L-arabinose is found throughout many different plant tissues in small amounts but strategically placed as side groups. ABF1 facilitates the breakdown of these side chains and cross-linking within the cell wall to work synergistically with other enzymes such as hemicellulases. This can increase the availability of fermentable sugars for biofuel production from biomass or pulp and paper production. This enzyme can increase the digestibility of livestock feed, has been used in the clarification of fruit juices and can aid in the aromatization of wines. Finally, ABF1 has possibilities as a food additive for diabetics due to the sweet taste and inhibitory effect on the digestion of sucrose75.
ABF1 was isolated from the fungus T. reesei and expressed in Escherichia coli in the pLD plasmid. The activity of this enzyme is assayed through the use of p-nitrophenyl-α-L-arabinofuranoside substrate in a 0.05M citrate buffer incubated at 50° C. for 10 minutes with enzyme crude extracts from plants or E. coli. The reaction is stopped with 1M Na2CO3 and the resultant p-nitrophenol is measured by spectrophotometer at 400 nm and compared to the positive standard (either p-nitrophenol or ABF, both of which are available commercially) to determine quantity. The pH and temperature optima studies are performed with the same substrate and measured in the same fashion 76. The activity of this enzyme has been known to be inhibited certain metal ions such as Cu2+, Hg2+, detergents and many chelating and reducing agents.75
Lignin Peroxidase (LipJ)
The enzyme Lignin peroxidase, commonly known as LipJ, remains novel and desirable for advancing the production of biofuel enzymes by means of transplastomic tobacco. Expression of LipJ should expand the spectra of exploitable biomass sources by hydrolysis of the inedible lignin and cellulose rich portions of biomass such as corn, sugarcane and wheat. Sources previously occluded as sources for biofuel due their high lignin content e.g. waste lumber, wood chips, peels from commercially prepared fruit and vegetables, could be hydrolyzed with LipJ. LipJ also advances biofuels production by ablating the intricate lignin structures within plant materials from inhibiting access to more valuable biomass substrates that provide valuable commercial, chemical and pharmaceutical products. These products are as of yet, limited in yield because of the protective nature of lignin surrounding them within biomass sources.
LipJ, was isolated from genomic DNA of Mycobacterium tuberculosis, expressed in a plasmid functional in both chloroplasts and E. coli. Three cultivars of transplastomic tobacco are expressing this vector in the primary selection round and await confirmation of chloroplast expression. Protein assays in E. coli have been designed for qualitative & quantitative analysis of LipJ expression, which is optimized for chloroplast derived LipJ.
Lipase Y (LipY), from Mycobacterium Tuberculosis, is a water soluble enzyme that catalyzes the hydrolysis of ester bonds and long-chain triacylglycerols, making it an ideal candidate for biofuel production. LipY is a membrane protein for Mycobacterium Tuberculosis and studies have already shown a humoral response will occur when LipY is combined with the serum of tuberculosis patients; therefore, LipY has the added benefit of being a potential vaccine for tuberculosis 77.
One simple assay for determining the activity of the cloned gene is a plate assay involving the fluorescent dye rhodamine B and a crude extract of lyses cells. If the gene was properly integrated and the protein folded correctly the assay will display an orange color when illuminated under a specific wavelength of light 80. Another assay measures the release of p-nitrophenol when p-nitrophenylstearate is incubated in various concentrations of E. coli or plant cell extract81.
Tobacco chloroplasts were transformed by microprojectile bombardment (biolistic transformation) as described before. Transgene integration was confirmed by PCR analysis using primers used in previous studies. Southern blot analysis was conducted to confirm homoplasmy. Tobacco (Nicotiana tabacum var. Petit Havana/LAMD/TN90) seeds were aseptically germinated on MSO medium in Petri dishes. Germinated seedlings were transferred to magenta boxes containing MSO medium. Leaves at 3-7 leaf stage of plant growth were cut and placed abaxial side up on a Whatman filter paper laying on RMOP medium in Petri plates (100×15 mm) for bombardment. Gold microprojectiles (0.6 μm) were coated with plasmid DNA (tobacco chloroplast expression vector) and biolistic mediated transformation were carried out with the biolistic device PDS1000/He (Bio-Rad) as published. After bombardment, leaves were incubated in dark for 48 hours to recover from damage. After 48 hours in dark, the bombarded leaves of Petit Havana (experimental cultivar) or LAMD and TN90 (commercial cultivar) were cut into 5 mm pieces and placed on plates (bombarded side in contact with medium) containing RMOP with 500, 150 and 200 mg/l of spectinomycin respectively for the first round of selection. After 4-5 weeks, resistant shoots will appear, whereas untransformed cells will die. Resistant shoots will be transferred to new RMOP-Spectinomycin plates and subjected to subsequent rounds of selection. The putative transplastomic shoots were confirmed by PCR and Southern analysis. Expression of cell wall degrading enzymes were confirmed by their respective assays and for those that antibody was available, Western blot analysis was performed.
The commercial cultivars yielded 40 metric tons biomass of fresh leaves as opposed to 2.2 tons in experimental cultivar Petit Havana. The commercial cultivars yield biomass 18 fold more than the experimental cultivar (Cramer et al., 1999). LAMD-609 is a low nicotine hybrid produced by backcrossing a Maryland type variety, MD-609, to a low nicotine-producing burley variety, LA Burley 21 (Collins et al., 1974), Tennessee 90 (TN 90) is a commercial cultivar used by Philip Morris (Lancaster Laboratories, PA). Both experimental cultivars LAMD and TN 90 were transformed with genes encoding biomass degrading enzymes. Although it is more challenging to transform these cultivars than the experimental cultivar, we succeeded in transforming both commercial cultivars with a number of genes encoding biomass degrading enzymes pectate lyases, cellulases, xylanases and endoglucanases. Transplastomic lines of commercial cultivars were homoplasmic, fertile and activities of expressed enzymes were similar to the experimental cultivar except that their biomass yield was much higher than Petit Havana. Table 8 shows enzymes that have been successfully introduced in plants, expressed and assayed for activity in experimental and commercial cultivars.
PCR was done using DNA isolated from leaf material of control and putative transgenic plants in order to distinguish true chloroplast transformants from nuclear transformants or mutants. Two separate PCR reactions was set up, one reaction checked for the integration of selectable marker gene into the chloroplast genome and the second checked integration of the transgene expression cassette. In order to test chloroplast integration of the transgenes, one primer (3M) will land on the aadA gene while another (3P) will land on the native chloroplast genome. No PCR product was obtained with nuclear transgenic plants or mutants using this set of primers. This screening is important for eliminating mutants and nuclear transformants. In order to conduct PCR analysis in transgenic plants, total DNA from unbombarded and transgenic plants was isolated as described by DNeasy plant mini kit (Qiagen). Integration of transgene expression cassette was tested using 5P/2M primer pair. Primer 5P lands in the aadA gene and 2M in the trnA, therefore, the PCR product showed whether the gene of interest had been introduced into the chloroplast genome via the homologous recombination process. A similar strategy has been used successfully by PI lab to confirm chloroplast integration of several foreign genes. The leaf pieces from PCR-positive shoots was further selected for a second round in order to achieve homoplasmy.
Southern blots were done to test homoplasmy. There are several thousand copies of the chloroplast genome present in each plant cell. When foreign genes are inserted into the chloroplast genome, not all chloroplasts will integrate foreign DNA resulting in heteroplasmy. To ensure that only the transformed genome exists in transgenic plants (homoplasmy), the selection process was continued. In order to confirm homoplasmy at the end of the selection cycle, total DNA from transgenic plants was probed with the radiolabeled chloroplast flanking sequences (the trnI-trnA fragment) used for homologous recombination. If wild type genomes are present (heteroplasmy), the native fragment size was observed along with transformed genomes. Presence of a large fragment due to the insertion of foreign genes within the flanking sequences and the absence of the native small fragment should confirm homoplasmy.
E. coli
The disclosures of the cited patent documents, publications and references, including those referenced in Tables II-VII, are incorporated herein in their entirety to the extent not inconsistent with the teachings herein. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
Aspergillus niger CBS
Aspergillus niger CBS
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus fumigatus
Aspergillus fumigatus
Bacillus licheniformis
Cryptococcus flavus
Trichoderma viride
Thermoascus aurantiacus
Agaricus bisporus
Thermobifida alba
Bacillus subtilis
Chaetomium cupreum
Paenibacillus polymyxa
Neocallimastix frontalis
Penicillium citrinum
Agaricus bisporus
Bacillus sp. (137)
Bacillus pumilus
Aeromonas punctata
Penicillium canescens
Cochliobolus carbonum
Aspergillus cf. niger
Bacillus alcalophilus
Trichoderma viride
Thermotoga maritima
Trichoderma viride
Gibberella zeae
Aeromonas caviae
Bacillus pumilus strain
Fusarium oxysporum f.
Fusarium oxysporum f.
Penicillium
purpurogenum
Streptomyces sp. S38
Bacillus sp. NBL420
Bacillus
stearothermophilus
Phanerochaete
chrysosporium strain
Thermoascus aurantiacus
Neocallimastix
patriciarum
Streptomyces avermitilis
Cochliobolus carbonum
Trichoderma reesei
Aspergillus tubingensis
Thermomonospora fusca
Aspergillus fumigatus
Aspergillus fumigatus
Aspergillus fumigatus
Aspergillus fumigatus
Vibrio sp. XY-214
Aspergillus niger
Bacteroides ovatus
Aspergillus clavatus
Aspergillus clavatus
Neosartorya fischeri
Aspergillus niger CBS
Aspergillus oryzae
Aspergillus oryzae
Vibrio sp. XY-214
Thermoanaerobacterium
Penicillium herquei
Bifidobacterium
adolescentis strain Int57
Aspergillus awamori
Bacillus pumilus IPO
Pyrus pyrifolia PpARF2
Clostridium stercorarium
Clostridium stercorarium
Aeromonas punctata
Clostridium stercorarium
Clostridium stercorarium
Talaromyces emersonii
Thermoanaerobacterium
Streptomyces
thermoviolaceus
Selenomonas
ruminantium
Bacillus pumilus
Bacillus
stearothermophilus
Azospirillum irakense
Cochliobolus carbonum
Streptomyces lividans
Bacillus sp. KK-1
Aspergllus nidulans
T. reesei
T. reesei
Clostridium stercorarium
Butyrivibrio fibrisolvens
Thermoanaerobacter
Paenibacillus sp. W-61
Aspergillus niger CBS
Aspergillus niger CBS
Aspergillus niger
Aspergillus niger
Aureobasidium pullulans
Geobacillus
stearothermophilus strain
Hypocrea jecorina strain
Aspergillus sojae
Penicillium
purpurogenum
Acremonium
cellulolyticus
Fusarium oxysporum f.
Clostridium stercorarium
Streptomyces
thermoviolaceus
Bifidobacterium longum
Aspergillus oryzae
Aspergillus oryzae
Clostridium
cellulovorans
Bacillus
stearothermophilus
Aspergillus oryzae
Aspergillus oryzae
Penicillium
purpurogenum
Cochliobolus carbonum
Cochliobolus carbonum
Cytophaga xylanolytica
Cytophaga xylanolytica
Clostridium stercorarium
Aspergillus niger
Neosartorya fischeri
Neosartorya fischeri
Neosartorya fischeri
Talaromyces stipitatus
Aspergillus niger
Aspergillus awamori
Penicillium
chrysogenum
Neurospora crassa
Volvariella volvacea
Aspergillus fumigatus
Aspergillus niger CBS
Neosartorya fischeri
A. niger
Didymella rabiei
Penicillium
purpurogenum
Aspergillus oryzae
esterase
Fibrobacter
succinogenes subsp.
succinogenes S85
Aspergillus ficuum
Bacillus pumilus
Trichoderma reesei
Thermoanaerobacterium
Streptomyces
thermoviolaceus
Streptomyces
thermoviolaceus
Streptomyces lividans
Orpinomyces sp. PC-2
Caldocellum
saccharolyticum
Talaromyces emersonii
Aspergillus niger
Aspergillus fumigatus
Aspergillus niger CBS
Aspergillus clavatus
Neosartorya fischeri
Neosartorya fischeri
Aureobasidium pullulans
Aspergillus tubingensis
Thermotoga maritima
Trichoderma reesei
Cellvibrio japonicus
Cellvibrio mixtus
Bacillus
stearothermophilus strain
Bacillus
stearothermophilus
Bacillus sp. TS-3
Bacillus subtilis
Aspergillus niger
Piromyces communis
Neocallimastix
patriciarum
Bacillus subtilis
Clostridium
thermocellum
Streptococcus equinus
Bacillus subtilis
Bacillus sp.
Clostridium
thermocellum
Bacillus circulans
Anaeromyces sp. W-98
Bacillus licheniformis
Orpinomyces sp. PC-2
Phanerochaete
chrysosporium
Alicyclobacillus
acidocaldarius
Clostridium
cellulolyticum H10
Aspergillus sulphureus
Phanerochaete
chrysosporium strain
Armillariella tabescens
Agaricus bisporus
Bacillus sp. JAMB750
Clostridium
thermocellum
Bacillus circulans isolate
Bacillus subtilis strain Z-2
Bacillus subtilis strain
Bacillus subtilis
Bacillus circulans isolate
Bacillus sp. JAMB-602
Agaricus bisporus
Piromyces sp.
Piromyces sp.
Piromyces sp.
Clostridium
thermocellum
Paecilomyces lilacinus
Bacillus circulans
Bacillus circulans
Dictyoglomus
thermophilum
Cellvibrio japonicus
Bacillus circulans
Bacillus
stearothermophilus
Orpinomyces sp. PC-2
Bacillus subtilis
Rhodothermus marinus
Bacillus subtilis
Thermotoga maritima
Cellulomonas fimi
Streptomyces lividans
Caldicellulosiruptor
saccharolyticus
Bacillus sp.
Bacillus circulans
Caldicellulosiruptor
saccharolyticus
Caldocellum
saccharolyticum
Aspergillus aculeatus
Trichoderma reesei
Bacillus sp.
Aspergillus niger CBS
Aspergillus niger CBS
Aspergillus clavatus
Neosartorya fischeri
Neosartorya fischeri
Aspergillus niger
Aspergillus niger
Aspergillus terreus
Emericella nidulans
Thermotoga neopolitana
Thermotoga neopolitana
Thermotoga maritima
Thermobifida fusca
Thermotoga maritima
Pyrococcus furiosus
Aspergillus aculeatus
Bacteroides fragilis
Bacteroides fragilis
Aspergillus fumigatus
Aspergillus fumigatus
Aspergillus niger CBS
Aspergillus niger CBS
Aspergillus niger (aglA)
Aspergillus niger CBS
Bacteroides
thetaiotaomicron
Bacteroides
thetaiotaomicron
Streptomyces avermitilis
Aspergillus clavatus
Bifidobacterium longum
Neosartorya fischeri
Aspergillus niger
Aspergillus niger
Lactobacillus fermentum
Emericella nidulans
Emericella nidulans
Pseudoalteromonas sp.
Lactobacillus plantarum
Bifidobacterium bifidum
Lachancea
thermotolerans
Lachancea
thermotolerans
Clostridium stercorarium
Bifidobacterium breve
Clostridium josui
Trichoderma reesei
Saccharomyces mikatae
Saccharomyces
paradoxus
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Penicillium
simplicissimum
Clostridium stercorarium
Bifidobacterium breve
Lactobacillus plantarum
Mycocladus
corymbiferus
Bacillus
stearothermophilus
Bacillus
stearothermophilus
Bacillus
stearothermophilus
Carnobacterium
piscicola
Thermus sp. T2
Phanerochaete
chrysosporium
Phanerochaete
chrysosporium
Zygosaccharomyces
mrakii
Thermus thermophilus
Torulaspora delbrueckii
Penicillium
purporogenum
Mortierella vinacea
Thermotoga neapolitana
Trichoderma reesei
Trichoderma reesei
Phlebia tremellosa
Phlebia tremellosa
Trametes versicolor
Trametes versicolor
Pleurotus pulmonarius
Phlebia radiate
Phlebia radiate
Trametes cervina
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Corynascus heterothallicus
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Clostridium cellulolyticum
Bacillus pumilus
Clostridium thermocellum
Ralstonia solanacearum
Ralstonia solanacearum
Cellulomonas fimi
Fibrobacter succinogenes
Bacillus sp. AC-1
Bacillus pumilus
Paenibacillus sp. KSM-N659
Paenibacillus sp. KSM-N440
Paenibacillus sp.
Paenibacillus sp.
Thermomonospora sp. MTCC 5117
Ralstonia solanacearum strain UW486
Bacillus licheniformis
Azoarcus sp. BH72
uncultured Butyrivibrio sp
Streptomyces halstedii
Clostridium thermocellum
Eubacterium cellulosolvens
Pectobacterium carotovorum (Erwinia
carotovora)
Cellulomonas fimi
Thermobifida fusca
Pectobacterium carotovorum
Clostridium cellulovorans
Ruminococcus albus
Ruminococcus albus
Myxococcus xanthus
Bacillus sp. BP-23
Ruminococcus flavefaciens
Anaerocellum thermophilum
Anaerocellum thermophilum
Pectobacterium carotovorum (Erwinia
carotovora)
Streptomyces rochei
Ruminococcus albus
Ruminococcus albus
Cellvibrio japonicus
Clostridium thermocellum
Clostridium thermocellum
Butyrivibrio fibrisolvens
Alicyclobacillus acidocaldarius
Xanthomonas campestris pv. campestris
Bacillus sp. BP-23
Bacillus licheniformis
Erwinia carotovora subsp. carotovora
Thermomonospora fusca
Alicyclobacillus acidocaldarius
Clostridium thermocellum
Clostridium thermocellum
Bacillus circulans
Bacillus amyloliquefaciens
Clostridium saccharobutylicum
Erwinia chrysanthemi
Clostridium saccharobutylicum
Pseudomonas sp. YD-15
Bacillus subtilis
Clostridium thermocellum
Clavibacter michiganensis subsp.
sepedonicus
Clostridium cellulovorans
Clostridium cellulovorans
Bacillus pumilus
Cellulomonas pachnodae
Clostridium thermocellum
Streptomyces halstedii
Bacillus sp
Clostridium longisporum
Bacillus agaradhaerens
Thermobifida fusca
Thermobifida fusca
Thermobifida fusca
Actinomyces sp. 40
Clostridium cellulovoran
Fibrobacter succinogenes
Ruminococcus flavefaciens FD-1
Erwinia chrysanthemi
Caldocellum saccharolyticum
Clostridium thermocellum
Clostridium cellulolyticum
Ruminococcus flavefaciens
Clostridium thermocellum
Erwina chrysanthemi
Caldocellum saccharolyticum
Streptomyces sp. M23
Clostridium thermocellum
Clostridium thermocellum
Clostridium thermocellum
Clostridium thermocellum
Cellulomonas fimi
Cellulomonas fimi
Piromyces rhizinflatus
Neocallimastix patriciarum
Penicillium decumbens
Polyporus arcularius
Polyporus arcularius
Fusicoccum sp. BCC4124
Thermomyces lanuginosus
Pleurotus ostreatus
Pleurotus ostreatus
Pleurotus ostreatus
Pleurotus ostreatus
Aspergillus fumigatus
Penicillium chrysogenum
Thermoascus aurantiacus
Aspergillus niger
Aspergillus niger
Trichoderma viride
Chaetomium thermophilum
Hypocrea koningii/Trichoderma koningii
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Trichoderma viride
Phanerochaete chrysosporium
Chaetomium thermophilum
Volvariella volvacea
Volvariella volvacea
Trichoderma reesei
Hypocrea koningii strain 3.2774
Hypocrea jecorina/Trichoderma reesei
Gibberella zeae
Fusarium venenatum
Gibberella zeae
Chaetomium thermophilum
Schizophyllum commune
Humicola grisea var. thermoidea
Agaricus bisporus
Agaricus bisporus
Phanerochaete chrysosporium
Thermoascus aurantiacus var. levisporus
Trichoderma parceramosum
Trichoderma parceramosum
Penicillium occitanis
Malbranchea cinnamomea
Stilbella annulata
Chaetomium thermophilum
Neocallimastix frontalis
Irpex lacteus
Trichoderma asperellum
Cochliobolus heterostrophus
Aspergillus aculeatus
Thermoascus aurantiacus
Trichoderma viride strain CICC 13038
Trichoderma viride
Piromyces sp. E2
Phanerochaete chrysosporium
Aspergillus sp.
Pseudoplectania nigrella
Orpinomyces sp. PC-2
Orpinomyces sp. PC-2
Talaromyces emersonii
Trichoderma viride
Geotrichum sp. M128
Aspergillus oryzae
Fusarium oxysporum
Talaromyces emersonii
Talaromyces emersonii
Talaromyces emersonii
Trichoderma koningii
Trichoderma koningii
Trichoderma koningii
Lentinula edodes
Phanerochaete chrysosporium
Agaricus bisporus
Phanerochaete chrysosporium
Pleurotus sajor-caju
Trichoderma harzianum
Orpinomyces sp. PC-2
Orpinomyces sp. PC-2
Hypocrea jecorina
Aspergillus niger
Aspergillus niger
Humicola grisea var. thermoidea
Humicola grisea
Trichoderma reesei
Trichoderma reesei
Pyrococcus horikoshii
Acidothermus cellulolyticus 11B; ATCC
Clostridium cellulolyticum
Clostridium thermocellum
Clostridium thermocellum
Clostridium thermocellum
Clostridium thermocellum
Bacillus subtilis
Bacillus lautus (strain PL236)
Cellulomonas fimi
Cellulomonas fimi
Thermobifida fusca
Thermomonospora fusca
Cellvibrio japonicus
Thermobispora bispora
Prevotella bryantii
Fibrobacter succinogenes
Pyrococcus furiosus
Thermococcus sp
Bacillus circulans
Bacillus sp. GL1
Bifidobacterium breve
Clostridium cellulovorans
Clostridium thermocellum
Thermobispora bispora
Paenibacillus sp. HC1
Sphingomonas paucimobilis
Thermoanaerobacter brockii
Thermotoga maritime
Thermus caldophilus
Thermotoga neapolitana
Thermus sp. Z-1
Butyrivibrio fibrisolvens
Cellvibrio gilvus
Flavobacterium meningosepticum
Erwinia chrysanthemi
Clostridium stercorarium
Paenibacillus sp.
Thermoanaerobacter brockii
Thermotoga maritime
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Piromyces sp. E2
Talaromyces emersonii
Aspergillus aculeatus
Emericella nidulans
Aspergillus niger
Aspergillus oryzae
Botryotinia fuckeliana
Dictyostelium discoideum
Coccidioides posadasii
Rhizomucor miehei
Thermoascus aurantiacus
Penicillium brasilianum
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
Rhizoctonia solani
Hypocrea jecorina/Trichoderma reesei
Penicillium occitanis
Chaetomium thermophilum
Penicillium brasilianum
Thermoascus aurantiacus
Aspergillus niger
Aspergillus clavatus
Aspergillus niger
Aspergillus oryzae
Aspergillus niger
Orpinomyces sp. PC-2
Hypocrea jecorina
Thermoascus aurantiacus
Thermoascus aurantiacus
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Chaetomium thermophilum
Aspergillus niger
Pichia anomala/Candida beverwijkiae
Hanseniaspora uvarum
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Hypocrea jecorina/Trichoderma reesei
Aspergillus fumigatus
Piromyces sp. E2
Aspergillus avenaceus
Phaeosphaeria avenaria
Phaeosphaeria nodorum
Phanerochaete chrysosporium
Candida albicans
Piromyces sp. E2
Trichoderma viride
Talaromyces emersonii
Debaryomyces hansenii/Candida famata
Debaryomyces hansenii/Candida famata
Acremonium cellulolyticus
Hypocrea jecorina/Trichoderma reesei
Humicola grisea var. thermoidea
Coccidioides posadasii
Coccidioides posadasii
Talaromyces emersonii
Coccidioides posadasii
Coccidioides posadasii
Coccidioides posadasii
Coccidioides posadasii
Aspergillus oryzae
Aspergillus niger
Candida wickerhamii
Candida wickerhamii
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Kluyveromyces marxianus
Pichia capsulata
Saccharomycopsis fibuligera
Saccharomycopsis fibuligera
Schizophyllum commune
Gibberella zeae
Schizosaccharomyces pombe
Schizosaccharomyces pombe
Schizosaccharomyces pombe
Hypocrea lixii
Thermomyces lanuginosus
Aspergillus niger strain F044
Antrodia cinnamomea
Aspergillus oryzae
Gibberella zeae
Gibberella zeae
Gibberella zeae
Aspergillus tamarii isolate FS132
Aureobasidium pullulans strain
Rhizopus microsporus var.
Fusarium oxysporum
Aspergillus tamarii isolate FS132
Neosartorya fischeri NRRL 181
Neosartorya fischeri NRRL 181
Nectria haematococca
Aspergillus terreus NIH2624
Galactomyces geotrichum
Yarrowia lipolytica
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Magnaporthe grisea
Penicillium expansum
Aspergillus niger
Aspergillus niger
Yarrowia lipolytica
Galactomyces geotrichum
Candida antarctica
Candida cylindracea
Yarrowia lipolytica
Yarrowia lipolytica
Rhizopus niveus
Galactomyces geotrichum
Rhizomucor miehei
Fusarium heterosporum
Candida albicans SC5314
Candida albicans SC5314
Rhizopus stolonifer
Candida albicans
Candida albicans
Candida albicans
Emericella nidulans
Gibberella zeae
Kurtzmanomyces sp. I-11
Candida deformans
Botryotinia fuckeliana
Penicillium allii
Aspergillus flavus
Aspergillus parasiticus
Yarrowia lipolytica
Candida parapsilosis
Penicillium expansum
Penicillium cyclopium
Pseudomonas sp.
Bacillus sp. NK13
Uncultured bacterium
Shewanella piezotolerans
Bacillus sp. Tosh
Bacillus subtilis strain FS321
Pseudomonas fluorescens strain
Pseudomonas fluorescens
Burkholderia cepacia
Burkholderia sp. HY-10
Pseudomonas aeruginosa
Pseudomonas fluorescens
Streptomyces fradiae
Geobacillus zalihae strain T1
Pseudomonas sp. MIS38
Uncultured bacterium
Burkholderia cepacia
Psychrobacter sp.
Bacillus subtilis strain Fs32b
Bacillus subtilis strain FS14-3a
Aeromonas hydrophila strain J-1
Acinetobacter sp. MBDD-4
Photorhabdus luminescens subsp.
akhurstii strain 1007-2
Uncultured bacterium clone
Geobacillus thermoleovorans
Bacillus pumilus strain F3
Pseudomonas fluorescens lipase
Geobacillus stearothermophilus
Serratia marcescens strain
Bacillus subtilis
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Uncultured bacterium clone pUE5
Serratia marcescens strain ES-2
Pseudomonas fluorescens strain
Stenotrophomonas maltophilia
Listonella anguillarum
Geobacillus sp.
Uncultured Pseudomonas sp.
Photobacterium sp. M37
Pseudomonas fluorescens
Pseudomonas aeruginosa
Bacillus pumilus mutant
Bacillus pumilus strain YZ02
Geobacillus thermoleovorans YN
Pseudomonas sp. CL-61
Burkholderia sp. 99-2-1
Burkholderia sp. MC16-3
Pseudomonas fluorescens
Geobacillus stearothermophilus
Burkholderia multivorans strain
Burkholderia multivorans strain
Bacillus pumilus
Bacillus sp. TP10A.1
Staphylococcus warneri
Staphylococcus warneri
Bacillus sp. L2
Bacillus megaterium
Pseudomonas aeruginosa
Bacillus sp. 42
Pseudomonas fluorescens
Burkholderia cepacia
Pseudomonas aeruginosa
Vibrio vulnificus
Uncultured bacterium plasmid
Thermoanaerobacter
tengcongensis
Pseudomonas aeruginosa
Staphylococcus epidermidis
Pseudomonas fluorescens
Pseudomonas fluorescens
Pseudomonas fluorescens
Pseudomonas fluorescens
Micrococcus sp. HL-2003
Pseudomonas sp. JZ-2003
Vibrio harveyi
Pseudomonas fluorescens
Bacillus sphaericus strain 205y
Bacillus subtilis
Synthetic construct
Serratia marcescens
Geobacillus thermoleovorans IHI-
Streptomyces rimosus
Pseudomonas luteola
Geobacillus thermoleovorans
Mycoplasma hyopneumoniae
Staphylococcus aureus
Bacillus sp. B26
Pseudomonas aeruginosa
Bacillus stearothermophilus
Bacillus stearothermophilus
Bacillus thermoleovorans
Bacillus stearothermophilus
Pseudomonas fluorescens
Pseudomonas sp. KB700A
Moritella marina
Staphylococcus xylosus
Staphylococcus warneri
Pseudomonas sp. UB48
Psychrobacter sp. St1
Pseudomonas fluorescens
Staphylococcus haemolyticus
Pseudomonas fluorescens
Pseudomonas aeruginosa
Streptomyces coelicolor
Pseudomonas fragi
Acinetobacter calcoaceticus
Streptomyces albus
Staphylococcus epidermidis
Pseudomonas sp. B11-1
Pseudomonas fluorescens
Pseudomonas aeruginosa
Pseudomonas wisconsinensis
Aeromonas hydrophila
Streptomyces sp.
Proteus vulgaris
Moraxella sp.
Serratia marcescens SM6
Staphylococcus aureus
Pseudomonas fluorescens
Bacillus subtilis
Staphylococcus epidermidis
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus amyloliquefaciens
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis 2633
Bacillus subtilis strain HA401
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Geobacillus stearothermophilus
Geobacillus stearothermophilus
Geobacillus stearothermophilus
Geobacillus (Bacillus)
stearothermophilus
Bacillus stearothermophilus
Bacillus stearothermophilus
Bacillus stearothermophilus
B. stearothermophilus
B. stearothermophilus
B. stearothermophilus
B. stearothermophilus
Bacillus stearothermophilus
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis strain RH
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
Bacillus licheniformis
B. licheniformis
B. licheniformis
Halothermothrix orenii
Xanthomonas campestris
Xanthomonas campestris pv.
campestris
Xanthomonas campestris pv.
campestris str. 8004
Xanthomonas campestris
Bifidobacterium adolescentis
Streptomyces lividans
Microbispora sp. V2
Streptomyces lividans
Thermoactinomyces vulgaris
Bacillus sp. YX
Bacillus sp. B1018
Bacillus halodurans
Bacillus sp. WHO
Bacillus sp. US149
Bacillus sp. KR-8104
Thermoactinomyces vulgaris
Bifidobacterium thermophilum
Clostridium sp.
Clostridium
thermoamylolyticum
Thermoactinomyces vulgaris R-47
Thermoanaerobacterium
thermosaccharolyticum
Chaetomium thermophilum
Aspergillus awamori GA I
Thermomyces lanuginosus
Aspergillus fumigatus
Rhizopus oryzae
Rhizopus oryzae
Aspergillus shirousami GLA
Talaromyces emersonii
Arxula adeninivorans
Aspergillus terreus NIH2624
Thermomyces lanuginosus
A. niger
Fomitopsis palustris
Aspergillus oryzae
Rhizopus oryzae
Saccharomycopsis fibuligera
Aspergillus awamori
Aspergillus oryzae
Trichoderma harzianum
Saccharomycopsis fibuligera
Amorphotheca resinae
Aspergillus niger
Saccharomycopsis fibuligera
Aspergillus niger strain
Aspergillus ficuum
Penicillium chrysogenum
Aspergillus niger
Aspergillus oryzae
Talaromyces emersonii
Aspergillus niger
Aspergillus kawachii
Candida albicans
Lentinula edodes
Aspergillus niger
Rhizopus oryzae
Corticium rolfsii
Aspergillus awamori
Saccharomyces cerevisiae
Aspergillus terreus
Debaryomyces occidentalis
Humicola grisea thermoidea
Saccharomyces diastaticus
Aspergillus shirousami
Fusicoccum sp. BCC4124
Cryptococcus flavus
Aspergillus awamori
Aspergillus awamori
Aspergillus awamori
Ajellomyces capsulatus
Phanerochaete chrysosporium
Paracoccidioides brasiliensis
Aspergillus fumigatus Af293
Aspergillus fumigatus Af293
Aspergillus fumigatus Af293
Aspergillus niger
Aspergillus nomius strain PT4
Aspergillus nomius strain KS13
Aspergillus nomius strain TK32
Aspergillus nomius
Aspergillus pseudotamarii
Aspergillus parasiticus
Aspergillus sp. BN8
Aspergillus flavus strain UR3
Aspergillus flavus strain AF70
Aspergillus clavatus NRRL
Aspergillus clavatus
Neosartorya fischeri NRR
Neosartorya fischeri
Ophiostoma floccosum
Ajellomyces capsulatus
Aspergillus oryzae
Lipomyces starkeyi
Cryptococcus neoformans var.
neoformans
Aspergillus oryzae
Candida albicans
Schwanniomyces occidentalis
Emericella nidulans
Lipomyces kononenkoae subsp.
spencermartinsiae
Aspergillus kawachii
A. awamori
Aspergillus clavatus NRRL 1
Aspergillus clavatus NRRL 1
Aspergillus oryzae
Bacillus sp. ZW2531-1
Geobacillus stearothermophilus
Bacillus sp. US149
B. acidopullulyticus
Bacillus licheniformis
Bacillus sp. WPD616
Bacillus stearothermophilus
B. stearothermophilus
Bacillus subtilis Bbma
Thermus sp. IM6501
Bacillus acidopullulyticus
Bacillus subtilis SUH4-2
Bacillus thermoalkalophilus
Geobacillus stearothermophilus
Hypsizygus marmoreus
Pleurotus eryngii
Coprinopsis cinerea strain
Pleurotus eryngii var. ferulae
Pleurotus eryngii var. ferulae
Sclerotinia minor
Crinipellis sp. RCK-1
Pholiota nameko strain Ph-5(3)
Trametes versicolor
Trametes versicolor
Coriolus versicolor
Thermus thermophilus
Bacillus halodurans
Colletotrichum gloeosporioides
Fusarium oxysporum
Neosartorya fischeri NRRL 181
Neosartorya fischeri NRRL 181
Pyrenopeziza brassicae
Botryotinia fuckeliana
Ascochyta rabiei
Aspergillus terreus NIH2624
Aspergillus terreus NIH2624
Aspergillus terreus NIH2624
Aspergillus terreus NIH2624
Aspergillus terreus NIH2624
Emericella nidulans
Emericella nidulans
Monilinia fructicola
Nectria haematococca
Nectria haematococca
Nectria ipomoeae
Phytophthora infestans clone
Phytophthora infestans
Phytophthora brassicae
Phytophthora brassicae
Phytophthora capsici
Monilinia fructicola
Blumeria graminis
Glomerella cingulata
Mycobacterium avium
Aspergillus oryzae
Fusarium solani
Fusarium solani pisi
Colletotrichum capsici
Alternaria brassicicola
Colletotrichum gloeosporioides
Colletotrichum gloeosporioides
Bacillus subtilis
Aspergillus fumigatus
Aspergillus fumigatus
Aspergillus niger
Aspergillus niger
Aspergillus niger
Aspergillus oryzae
Pseudoalteromonas haloplanktis
Penicillium griseoroseum
Penicillium griseoroseum
Aspergillus niger
Aspergillus niger
Aspergillus niger
Mycosphaerella pinodes
Aspergillus niger
Aspergillus niger
Emericella nidulans
Emericella nidulans
Erwinia chrysanthemi
Erwinia sp. BTC105
Erwinia chrysanthemi
Erwinia carotovora
Bacillus sp.
Erwinia chrysanthemi
Erwinia chrysanthemi
Bacillus halodurans strain ATCC
Aspergillus niger
Aspergillus niger
Penicillium expansum
Blumeria graminis f. sp. tritici
Aspergillus oryzae
Aspergillus oryzae
Colletotrichum gloeosporioides
Colletotrichum gloeosporioides
Bacillus sp. P-358
Aspergillus niger
Erwinia carotovora
Erwinia carotovora
Bacillus sp. YA-14
Streptomyces thermocarboxydus
Pseudomonas viridiflava strain
Pseudomonas viridiflava strain
Pseudomonas viridiflava strain
Pseudomonas viridiflava strain
Aspergillus fumigatus Af293
Aspergillus niger CBS 513.88
Emericella nidulans
Bacillus sp. P-4-N
Bacillus sp. P-4-N
Fusarium solani
Fusarium solani
Fusarium solani
Fusarium solani pisi (Nectria
hematococca)
This application is related to U.S. Provisional Patent Application No. 61/032,536 filed Feb. 29, 2008 to which priority is claimed under 35 USC 119 and is incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61032536 | Feb 2008 | US |
