Patent Application
20060005286
The instant invention pertains to the production of enzymes of interest in the seeds of transgenic plants and the use of the thus-produced seeds in industrial processes, without the need for extraction and/or isolation of th enzyme.
A number of industries are using enzymes for their processes. These include detergents, textiles, dairy, food and beverage, feed and other industries.
At the present, enzymes are produced on an industrial scale by fermentation processes or are isolated from plant or animal sources. Microbially produced enzymes include proteases, amylases, cellulases, pectinases, phytases and others. Enzyme production by a fermentation process is highly efficient and production levels of more than 10 grams per liter culture medium can be reached.
The possibility of using transgenic plants as a production system for valuable proteins has been proposed. Examples to date are the production of interferon in tobacco (Goodman et al., 1987), enkephalins in tobacco, Brassica napus and Arabidopsis thaliana (Vandekerckhove et al. , 1989), antibodies in tobacco (Hiatt et al., 1990) and human serum albumin in tobacco and potato (Sijmons et al., 1990).
In practice, the transformation of an increasing number of plant species, especially dicotyledonous species (e.g. tobacco, potato, tomato, Petunia, Brassica), has become a routine procedure for workers skilled in the art (Klee et al., 1987; Gasser & Fraley, 1989). Strategies for the expression of foreign genes in plants have become well established (Gasser & Fraley, 1989). Regulatory sequences from plant genes have been identified that are used for the construction of chimeric genes that can be functionally expressed in plants and plant cells.
For the introduction of gene constructions into plants, several technologies are available, such as transformation with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Using this strategy, a wide variety of plant tissues have been exploited, the choice being largely dependent on the plant species and its amenability in tissue culture. Successful examples are the transformation of protoplasts, microspores or pollen, and explants such as leaves, stems, roots, hypocotyls and cotyls. Furthermore, methods for direct DNA introduction in protoplasts and plant cells or tissues are used such as microinjection, electroporation, particle bombardment and direct DNA uptake (Gasser & Fraley, 1989).
Proteins may be produced in plant seeds using a variety of expression systems. For instance, the use of a constitutive promoter such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley et al., 1982) will result in the accumulation of the expressed protein in the seeds, inter alia, of the transgenic plant. Alternatively, use may be made of promoters from genes encoding seed storage proteins. Seed storage proteins are expressed in a highly tissue-specific and stage-specific manner (Higgins, 1984; Shotwell & Larkins, 1989), i.e., the genes are expressed only in seed and only during the stage of seed development.
A seed storage protein (reviewed in Higgins. 1984; Shotwell & Larkins, 1989) is defined as any protein which accumulates in significant quantities (up to 90% of total seed protein) in the developing seed and which on germination is hydrolyzed to provide a nutritional source for the early stages of seedling growth. The proteins are contained in an intracellular compartment called the protein body or storage vacuole. This protein body contains protease inhibitors and creates a protease-free environment. The proteases that degrade the storage proteins become active 3-6 days after germination (Larkins, 1981).
Many seed storage protein genes have been isolated and characterized, as well as their 5′ and 3′ flanking regions (reviewed by Casey & Domoney, 1989). Examples for the globulins and albumins are the glycinin and conglycinin genes of soybean (Fischer & Goldberg, 1982; Harada et al., 1989), the legumin and vicilin genes from pea (Lycett et al., 1984; Higgins et al., 1988), the 11S field bean gene (Baumlein et al., 1986), the 7S phaseolin gene from Phaseolus (Doyle et al., 1986), the cruciferin and napin genes from Brassica (Ryan et al., 1989; Scofield & Crough, 1987; Radke et al., 1988), the helianthin gene from sunflower (Vonder Haar et al., 1988: Jordano et al., 1989) and the 2S albumin and cruciferin genes from Arabidopsis thaliana (Vandekerckhove et al., 1989; Pang et al., 1988). other examples may be found in the genes encoding the prolamins and glutelins (Casey & Domoney, 1987). Generally, the storage proteins are encoded by multigene families.
Seed storage protein genes have been transferred to tobacco, petunia and rapeseed (Okamura et al., 1986: Beachy et al., 1984; Sengupta-Gopalan et al., 1995; Higgins et al., 1989; Ellis et al., 1988; Barker et al., 1988, Vandekerckhove et al., 1989; Altenbach et al., 1989). The 5′ upstream regulatory region of beta-phaseolin from pea was used to direct the expression of beta-glucoronidase (Bustos et al., 1989), phytohemaglutinin (Voelker et al., 1989), luciferase (Riggs et al., 1989) and zein (Hoffman et al., 1987) in tobacco. The promoter of the Arabidopsis thaliana 2S albumin gene was used to direct the expression of a modified 2S albumin from the same species in tobacco, Brassica napus and Arabidopsis thaliana (Vandekerckhove et al., 1989). The genes mentioned above were expressed in a tissue-specific and developmentally regulated manner, i.e., in seed during seed development. The expression levels in all these reports varied, but reached levels as high as 1.7% of the total seed protein (Voelker et al., 1989). It has been found that cDNA car replace genomic DNA containing introns as the basis for obtaining a functional and stable mRNA in the heterologous expression (Chee et al., 1986). These results demonstrate that a person skilled in the art of plant molecular biology can design strategies for seed-specific expression of a given gene in a target plant species that is amenable to transformation technology.
During seed development of dicots, a large part of the total protein synthesis is directed into the vacuole or the protein bodies of storage parenchyma cells. For regulation of this process, the proteins are generally synthesized as precursors. The precursor proteins are equipped with hydrophobic signal peptides, usually at the N-terminus, that are cleaved off at specific stages. A large number of storage protein signal peptides have been described (Doyle et al., 1986; Pang et al., 1988; Vonder Haar et al., 1988; Iturriaga et al., 1989; Dorel et al., 1989; Voelker et al., 1989; Hattori et al., 1985; Lycett et al., 1983; Smith & Raikhel, 1989).
The general applicability of signal peptides in heterologous expressions systems (e.g., Sijmons et al., 1990; Vitale & Bollini, 1986; Slightom et al. 1986; Della-Cioppa et al., 1987) seems to support the idea that a fusion of a signal peptide with a heterologous “passenger protein” may be used for transporting and processing of the passenger protein. The references suggest that a variety of potential “passenger proteins” are candidates for such an expression system.
However, in spite of the attractiveness and viability of the use of plants as bioreactors, the system up until now is not without difficulties. For the examples described above, the plant is used as a bioreactor and the protein of interest is then isolated from the transgenic plant material, i.e., from the tissues containing the highest levels of the protein of interest. The isolation of the protein of interest from the seeds in which it is produced inherently introduces complications as well as added cost (Krebbers & Vandekerckhove. 1990).
A possible solution to this problem may be to avoid the need to extract the expressed protein from the plant material. East German patent DD 275,704 discloses a construct for the expression of a heat stable beta-glucanase in the ungerminated seeds of transformed barley plants and the use of the seeds in brewing processes. However, a persistent problem in the manipulation of small grain cereal crops has been not only the transformation of the protoplasts of cereal plants but the regeneration of the transformed plants as well, which are not enabled in the patent's disclosure. Thus, it would not be possible to obtain enzyme-containing seeds using the process as described in the publication.
According to the present invention, seeds containing at least one enzyme of interest are provided, which can be used in food and feedstuffs such as dough or other bakery products as catalysts and for digestive reactions, without the need for first extracting and/or isolating said enzymes.
DNA constructs are provided for the transformation of plants which are, in turn, capable of expression of a variety of enzymes of interest in seeds. The constructs employ signal sequences operably linked to a DNA sequence encoding the desired enzyme to be expressed. Such constructs are under the control of regulatory sequences which are capable of directing the expression of the desired enzymes in seeds.
The present invention also provides for the use of the seeds of transgenic plants as a stable and manageable storage form of enzymes. The enzymes are maintained in a dessicated environment which is low in protease activity and thus are protected against degradation.
Moreover, the use of seeds for the storage of enzymes provides a stable vehicle which is easily packaged and transported, and easily handled during actual use.
The present invention further provides a viable solution to the expensive and problematic process of the extraction of enzymes of interest from the seeds in which they are produced. The enzymes remain stable inside the seed and as such may be used as a replacement for the pure enzyme. This benefit, coupled with the low cost of growing seed-producing plants, provides an economical source of such enzymes. Thus, the present invention allows for the reduction of costs associated with the production, storage and use of a variety of enzymes.
Enzymes of interest which nay be produced by the present invention include any enzymes which are capable of use in industrial and food processes such as bakery processes.
The enzymes of interest include enzymes which are heterologous to the plant (i.e. not native to the plant species) in which they are produced. Also intended are enzymes, homologous to the plants (i.e. native to the plant species) in which whey are produced, which are overexpressed via recombinant DNA techniques.
Such enzymes are selected from hydrolases such as proteases, cellulases, hemi-cellulases, phosphatases, lipases, pectinases, amylases, lysozymes, pullulanases and chitinases; lyases such as pectinlyase; isomerases such as glucose isomerase; glucose transferases such as cyclodextrin glycosyl- transferase; and oxygenases such as lipoxygenase.
Preferred enzymes are α-amylase, cellobiohydrolase, cyclodextrin glucanotransferase, endo-glucanase, endo-xylanase, endo-galactanase, lipoxygenase, α-galactosidase, hexose oxidase, arabinanase, protein disulfide isomerase, serine-proteases, chymosin, papain, gastric lipases, pectin lyase and glucose isomerase.
By industrial processes is intended processes in which extracted and/or isolated enzymes are normally included in a reaction mixture, either in vivo or in vitro, containing at least one substrate, wherein the enzymes are capable of catalyzing such reaction of the substrate(s) so as to produce desired effects or products.
Examples of such industrial processes include, but are not limited to, in vitro processes such as the use of phytases in soy processing or in an industrial process such as wet milling or for the production of inositol or inositol-phosphates from phytate. Hemi-cellulases and cellulases may also be used as cell wall degrading enzymes, in general. In a like manner, α-amylases and other enzymes such as xylanases, glucosyl transferases, isomerases and oxygenases may be used in the baking industry to improve the consistency of baked products; α-amylase, amyloglucosidase, xylanases and/or glucose isomerase may be used in starch liquefaction; ligninases and/or xylanases may be used in the paper industry: glucanases, pectinases and/or cellulases may be used in the food and beverage industry, e.g., in fermentation or brewing processes.
Apart from the above-mentioned action of enzymes in in vitro processes, the enzymes stored in seeds may be used to catalyze digestive reactions in vivo. In this manner, the enzymes facilitate the release of nutrients from foodstuffs which otherwise would not be readily available to the animal which ingests them.
Enzymes to be used in such in vivo processes include, but are not limited to, phytases, cellulases, hemi-cellulases, pectinases and amylases. The enzymes as will lead to improved digestion of the foodstuffs. Enzymes as digestive aids can also be used in humans, for instance in illnesses such as cystic fibrosis or other causes of pancreatic insufficiencies. Lipases, proteases, and cellulases contained in seeds may be used as therapeutic additives to alleviate digestive problems associated with such illnesses.
According to the present invention, the desired enzyme is produced in the seeds of transgenic plants. The thus-produced seeds are in turn used in an industrial process which requires the enzyme contained therein, without the need for first extracting and/or isolating the enzyme.
Bakery enzymes can be defined as enzymes especially useful in bakery processes. Such enzymes are, for instance, protein disulfide isomerase, which through a process of breaking and formation of cys-cys bonds in proteins gives improved rheological properties of the dough, more relaxed and better machinability of doughs and a larger volume to the baked product; lipoxygenase-3 (LOX-3) which added to a bread dough results in bleaching of endogenous carotenoids which is desirable in the baking process of white bread and, in addition, strengthens the dough by its oxidizing capacity; cyclodextrin glucano-transferase (CGTase) which when added to a bread dough results in an increase in volume of the baked product and softening of the bread crumb and (endo-)xylanase which results in better dough stability, improved crumb structure and also increases the volume of the baked product.
It will be appreciated by those skilled in the art that seeds containing enzymes for industrial use may be directly used in such processes, or may first be prepared for such use by means of grinding to the desired consistency. In either case, the whole or ground seeds may be fed as such into the desired process without the need for further extraction and/or isolation of the enzyme, and also without loss of activity of the enzyme.
Transgenic plants, as defined in the context- of the present invention, include plants and their progeny, which have been genetically modified to cause or enhance production of at least one enzyme of interest in their seeds. The production of the enzymes of interest is thus increased over the level found in the wild-type plant variety.
In the context of the present invention. the phrase “seeds containing an enhanced amount of enzyme” refers specifically to a statistically significant number of seeds which, on average, contain a statistically significant greater amount of an enzyme as compared with the average amount of enzyme in an equal number of non-modified seeds.
Plant genera which are capable of producing the enzyme of interest in their seed by practice of the present invention include, but are not limited to, i Nicotiana (e.g., tabacum), Brassica (e.g., napus and oleracea), Arabidopsis, Glycine (e.g., max), Zea (e.g., mays), Amaranthus, Hordeum (e.g., vulgarum), and Pisum (e.g., sativum) Juglans (e.g. regia), Arachis (e.g. hypoceae), Medicago (e.g. sativa), Phaseolus (e.g. vulgaris). Pisum (e.g. sativum), Triticum (e.g. aestivum) Panicum L., Helianthus (e.g. annus), Avena (e.g. sativa) and Oryza (e.g. sativa).
Preferably, the species of choice must have a large production of seed per plant per annum and the chemical and physical properties of the seed should be compatible with the industrial process for which the enzyme is produced. For instance, in some cases when these seeds (after transformation of the parent plant) are to be included in foodstuffs, one may choose a plant species that produces seeds which are low in tannins or other antinutritional factors. In other cases the ability of the transgenic seeds to be ground to the desired consistence may be the criterion of choice when the seeds are used as additives, e.g., in flour. In yet another embodiment, the seeds containing the enzymes of interest may be directly applied to the desired process (e.g. in feedstuffs), per se, optionally preceded by dehusking and/or drying after harvesting.
The choice of the most suitable plant species can be based on reconstitution experiments. The enzyme of interest can be added together with wild-type seeds to the industrial process for which the transgenic seeds will eventually be produced. In bakery processes seeds which qualify to be added to the dough are seeds of plants which are normally used in baking processes, such as wheat, rye, rice, sunflower, barley, maize and poppy seeds. However, it is also possible to add small amounts of seeds of other planes such as rapeseed.
The genetic modification of the above-described seed-producing plants intends that an expression construct containing a gene encoding an enzyme of interest is introduced into the target plant. This expression construct may include a gene, heterologous to the plant, which is under the control of promoter and termination regions capable of regulating the expression of the gene encoding the enzyme of interest in the seeds of the plant. Also intended is a gene, homologous to the plant which as under the control of a regulatory region capable of effecting the overproduction of the enzyme of interest. By overproduction is intended production of the enzyme of interest at levels above that which is found in the wild-type plant variety.
Transformation of plant species is now routine for an impressive number of plant species. including both the Dicotyledoneae as well as the Monocotyledoneae. In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell. Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol. S. 363-373), electroporation of protoplasts (Shillito R. D. et al., 1985 Bio/Technol. 3, 1099-1102). microinjection into plant material (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), (DNA or RNA-coated) particle bombardment of various plant material (Klein T. M. et al., 1987, Nature 327, 70), infection with (non-integrative) viruses, in planta Agrobacterium tumefaciens mediated gene transfer by infiltration of adult plants or transformation of mature pollen or microspores (EP 0 301 316) and the like. A preferred method according to the invention comprises Agrobacterium-mediated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Pat. No. 4,940,838).
Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material. Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or (tissue) electroporation (Shimamoto, et al, 1989, Nature 338, 274-276). Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2, 603-618). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee. 1989, Plant Mol. Biol. 13, 21-30). Wheat plants have been regenerated from embryogenic suspension culture by selecting embryogenic callus for the establishment of the embryogenic suspension cultures (Vasil, 1990 Bio/Technol. 8, 429-434). The combination with transformation systems for these crops enables the application of the present invention to monocots.
Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains (vide WO 94/00977; EP 0 159 418; Gould J. Michael D, Hasegawa O, Ulian EC, Peterson G. Smith R H. (1991) Plant Physiol. 95, 426-434).
It is known that practically all plants can be regenerated from cultured cells or tissues. The means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Shoots may be induced directly, or indirectly from callus via organogenesis or embryogenesis and subsequently rooted. Next to the selectable marker, the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotvpe and on the history of the culture. If these three variables are controlled regeneration is usually reproducable and repeatable.
The expression of recombinant genes in plants involves such details as transcription of the gene by plant polymerases, translation of mRNA, etc., which are known to persons skilled in the art of recombinant DNA techniques. Only details relevant for the proper understanding of this invention are discussed below.
Regulatory sequences which are known or are found to cause sufficiently high expression (for the purpose of the specific application, as discussed below) of the recombinant DNA in seeds, can be used in the present invention. Such regulatory sequences may be obtained from plants or plant viruses, or chemically synthesized. Such regulatory sequences are promoters active in directing transcription in seeds. These include, but are not limited to, promoters from seed-specific genes, especially those of storage protein genes, such as the cruA promoter of Brassica napus (Ryan et al., 1989) or promoters of constitutively expressed genes, such as the 35S promoter of CaMV (Cauliflower Mosaic Virus) (Guilley et al., 1982). Other regulatory sequences are terminator sequences and polyadenylation signals, including every sequence functioning as such in plants; examples are the 3′ flanking region of the nopaline synthase gene of Agrobacterium tumefaciens or the 3′ flanking region of the cruA gene of Brassica napus. The regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV, and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Virus (AlMV) RNA4 (Brederode et al., 1980) or any other sequences functioning as such.
The protein of interest should be in an environment that allows optimal stability of the protein during seed maturation. The choice of cellular compartments, such as cytosol, endoplasmic reticulum, vacuole, protein body or periplasmic space can be used in the present invention to create such a stable environment, depending on the biophysical parameters of the protein of interest. Such parameters include, but are not limited to, pH-optimum, sensitivity to proteases or sensitivity to the molarity of the preferred compartment. Although homologous signal sequences are preferred, heterologous signal sequences may be used as well. Especially preferred are signal sequences obtained from seed storage proteins.
The seed storage proteins can be divided into four major classes based on solubility characteristics:
1. Albumins—soluble in water and subdivided in two main classes (12S and 2S). The 12S class includes lectins isolated from pea and various beans, e.g., 2S albumins from Brassica napus, Arabidopsis thaliana, Ricinus communis (castor bean), Bertholletia excelsa (Brazil nut), pea, radish and sunflower.
2. Globulins-soluble in salt solutions and may be either of the 7-8S class like the phaseolins from Phaseolus, the vicilins from pea, the conglycinins from soybean, the oat-vicilins from oat and 7S globulins from other species, or of the 11-14S class such as the legumins from pea, the glycinins from soybean, the helianthins from sunflower, the cruciferins from rape or 11-14S proteins from other species such as Arabidopsis and bean.
3. Prolamins—soluble in aqueous alcohol, e.g., zeins from corn, the hordeins from barley, the gliadins isolated from wheat and the kafirins from sorghum.
4. Glutelins—soluble in acidic or alkaline solutions and may be isolated from wheat.
Although there are exceptions, the major storage proteins in seed of dicotyledonous plants are globulins, and those of monocotyledonous plants are prolamins and glutelins.
All parts of the relevant DNA constructs (promoters; regulatory, stabilizing, signal or termination sequences) of the present invention may be modified, if desired, to affect their control characteristics using methods known to those skilled in the art. The amount of recombinant protein (the “expression level”) needed in the seed should be sufficiently high to use the transgenic seed as a minor additive (on a volume, weight or cost base) in all preferred embodiments of the present invention.
To obtain transgenic plants capable of constitutively expressing more than one chimeric gene, a number of alternatives are available including the following:
A. The use of DNA, e.g a T-DNA on a binary plasmid, with a number of modified genes coding for the enzymes of interest physically coupled to a second selectable marker gene. The advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus.
B. Cross-pollination of transgenic plants each already capable of expressing one or more enzymes of interest, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coding for enzymes of interest coupled to another selectable marker. Afterwards the seed, which is obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the enzymes themselves. The plants obtained from the selected seeds can afterwards be used for farther crossing. In principle the chimeric genes are not on a single locus and the genes may therefore segregate as independent loci.
C. The use of a number of a plurality chimeric DNA molecules, e.g. plasmids, each having one or more chimeric genes coding for an enzyme of interest and a selectable marker. If the frequency of co-transformation is high, then selection on the basis of only one marker is sufficient. In other cases, the selection on the basis of more than one marker is preferred.
D. Consecutive transformation of transgenic plants already containing a first, second, (etc), chimeric gene with new chimeric DNA, optionally comprising a selectable marker gene. As in method B, the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci.
E. Combinations of the Above Mentioned Strategies.
The actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids) but is not critical with respect to the described invention.
After stable incorporation of the transformed gene sequences into the transgenic plants, the traits conferred by them can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
It is pointed out that seeds containing enhanced amounts of enzymes could be obtained by processes, known to those skilled in the art, other than those recombinant processes mentioned above provided those processes result in obtaining seeds with enhanced amounts of enzymes as compared with the wild-type seeds. For example, it might be possible to obtain such seeds by the use of somaclonal variation techniques. Further, such techniques could be used by themselves or in combination with breeding techniques which employ the concept of cytoplasmic male sterility (Cms) or nuclear male sterility (Nms) (Mariani et al., 1990). Techniques such as somaclonal variation and cross-breeding involving the use of Cms or Nms could be used in combination with the recombinant technologies mentioned above in order to further increase the relative amounts of enzymes present within the seeds. With respect to non recombinant techniques which might be utilized to enhance the amount of enzymes within seeds reference is made to U.S. Pat. No. 4,378,655 issued 7 Apr. 1933 which patent is incorporated herein by reference to disclose such techniques. It is pointed out that there are numerous publications describing breeding techniques involving cytoplasmic male sterility which was discovered by P. LeClercq in 1968 and the corresponding dominant fertility restoring genes (Rf) which were discovered by M. L. Kinmar et al. in 1970. Recently, the use of nuclear male sterility has been described by Mariani et al. in 1990. More generalized disclosures relating to plant breeding are discussed within James R. Welsh “Fundamentals of Plant Genetics and Breeding”, 1981 as well as within J. M. Poehlman, “Breeding Field Crops”, 1959.
In one embodiment of the present invention, a genomic DNA fragment encoding α-amylase from Bacillus licheniformis is placed under the control of the CaMV 35S promoter and enhancer sequences. The mRNA stabilizing leader sequence of RNA4 from AlMV is included, as well as the terminator and polyadenylation signal sequences of the nopaline synthase gene of Agrobacterium tumefaciens. The entire construct is thereafter subcloned into a binary vector. This vector is introduced into Agrobacterium tumefaciens which contains a disarmed Ti plasmid. Bacterial cells containing this construct are cocultivated with tissues from tobacco plants, and transformed plant cells are selected by nutrient media containing antibiotics and induced to regenerate into differentiated plants on such media. The resulting plants produce seeds that contain and express the DNA construct.
The α-amylase enzyme activity of the transgenic seeds may be determined via methods, not critical to the present invention, such as direct enzyme assays using colorimetric techniques or native gel assays.
The seeds can be used as a source of α-amylase, which can be directly used in industrial processes, such as bakery. Preferably, the seeds are first ground and the entire (ground) mixture may be used in the bakery process, as may be determined by one of ordinary skill in the art.
The following examples are provided so as a to give those of ordinary skill in the art a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, pH, etc.) but some experimental errors and deviation should be accounted for. Unless indicated otherwise, temperature is in degrees Centrigrade and pressure is at or near atmospheric.
In this example, the construction of the binary vector pMOG23 (in E. coli K12 DH5α, deposited at the Centraal Bureau voor Schimmelcultures on Jan. 29, 1990 under accession number CBS 102.90) is described.
The binary vector pMOG23 (
First, the positions of the left border (LB) and the right border (RB) are switched with reference to the neomycine phosphotransferase gene II (NPTII gene). Secondly, the orientation of the NPTII gene is reversed giving transcription in the direction of LB. Finally, the polylinker of Bin19 is replaced by a polylinker with the following restriction enzyme recognition sites: EcoRI, KpnI, SmaI, BamHI, XbaI, SacI, XhoI, and HindIII.
An expression construct is constructed in such a way that seed-specific expression is obtained, using sequences of the Brassica napus 12S storage protein gene cruciferin (cruA; Ryan et al., 1989). These sequences may be replaced by those from similar seed-specific genes to achieve the same goal as is the objective of this invention.
For all E. coli transformations in this example, E. coli K-12 strain DH5αis used.
a) Construction of the Expression Construct
For the construction of the expression construct for seed-specific expression, the promoter and terminator sequences from the cruciferin A (cruA) gene of Brassica napus cv. Jet Neuf are synthesized using PCR technology with isolated genomic DNA (Mettler, I. J., 1987) as a template. This gene shows seed-specific expression and its coding and flanking sequences have been determined (Ryan et al., 1989).
Two sets of oligonucleotides are synthesized. One to allow amplification of the cruA 5′ flanking region and part of the signal peptide encoding sequence as an EcoRI/NcoI fragment:
5′ GTTCGGAATTCGGGTTCCGG 3′ and 5′ AACTGTTGAGCTGTAGAGCC 3′.
The other for amplification of the 3′ flanking sequence as a BglII/HindIII fragment:
5′ CTTAAGATCTTACCCAGTGA 3′ and 5′ CGGAGAAGCTIGCATCTCGT 3′.
The oligo's are designed to contain suitable restriction sites at their termini to allow direct assembly of the expression construct after digestion of the fragments with the restriction enzymes.
The 5′ fragment of the cruA gene, that includes 54 nucleotides of the sequence encoding the signal peptide is cloned into vector pMOG445 (Oligonucleotide duplex E (
In this vector the gene of interest can be cloned. The resulting vector is mobilized, in a triparental mating with the E. coli K-12 strain RK2013 (containing plasmid pRK2013) (Ditta et al., supra), into Agrobacterium strain LBA4404 (Hoekema et al., 1983, supra) which contains a plasmid with the virulence genes necessary for T-DNA tranfer to the plant.
In this example, the α-amylase gene from Bacillus licheniformis is tailored and cloned in an expression cassette for constitutive expression which also contains the coding information for a signal peptide sequence of plant origin. As a final step, the entire construct is cloned in a binary vector, transferred to Agrobacterium tumefaciens strain LBA4404, which is used to transform the plant of interest. Any other gene or cDNA may be cloned in a similar way as is described here for the α-amylase gene.
All transformations in this example are done in E. coli K-12 strain DH5 -α.
a) Tailoring of the α-amylase Gene of Bacillus licheniformis
The α-amylase gene from Bacillus lichenformis (
b) Construction of Vector pMOG29
The expression construct of ROK1 (Baulcombe et al., 1986) is cloned as an EcoRI/HindIII fragment into pUC18. This construct contains the Cauliflower Mosaic Virus (CaMV) 35S promoter on an EcoRI/BamHI fragment and the nopaline synthase (nos) transcription terminator on a BamHI/HindIII fragment. The promoter fragment consists of the sequence from −800 to +1 of the CaMV 35S promoter. Position +1, which is included: is the transcription initiation site (Guilley et al., 1982). The sequence upstream of the NcoI site at position −512 is deleted and this site is changed into an EcoRI site. This is done by cutting the expression construct present in pUC18 with NcoI, filling in the single-stranded ends with Klenow polymerase and ligation of an EcoRI linker. The resulting plasmid is cut with EcoRI, resulting in the deletion of the EcoRI fragment carrying the sequences of the 35S promoter upstream of the original NcoI site. The BamHI/HindIII fragment, containing the nos terminator is replaced by a synthetic DNA fragment (oligonucleotide duplex A,
c) Cloning of the α-amylase Gene from Bacillus licheniformis in the Binary Vector
Plasmid pMOG320 is digested with HgaI and BamHI. The HgaI/BamHI fragment, which encodes mature α-amylase from amino acid 9 onward, is cloned in a three-way ligation with the synthetic oligonucleotide duplex F (
In this example tobacco is transformed by cocultivation of plant tissue with Agrobacterium tumefaciens, containing a binary vector with the chimeric α-amylase gene. Transgenic plants are selected on antibiotic resistance. The seeds of the transgenic plants are assayed for α-amylase activity. High expressors are analyzed more thoroughly and used in further experiments.
Agrobacterium strain LBA4404 (pMOG227) is used for transformation experiments. Transformation of tobacco (Nicotiana tabacum SR1) is carried out using cocultivation of leaft discs according to the procedure of Morsch et al., (1985). Transgenic plants are regenerated from shoots that grow on selection medium (100 mg/l kanamycin). Young plants are assayed for NPTII-activity, grown to maturity and allowed to self-pollenate and set seed. Seeds from individual transformants are pooled and part of the seed sample is assayed for the presence of α-amylase. From clones with the highest expression levels, compared to untransformed control seeds, the remaining seeds are germinated on kanamycin (200 mg/L) (hence also transgenic for α-amylase) and selected and used for mass propagation of plants capable of producing seeds containing the highest amounts of α-amylase. A maximum α-amylase expression level of 0.4% of the total soluble seed protein was observed. These seeds can then be used, e.g. for digestion experiments.
Bacillus licheniformis α-amylase, expressed in tobacco seed, was applied in the liquefaction of starch as follows: 100 grams of both α-amylase-expressing and control tobacco seeds are harvested. Seeds were ground with a sieve (Retch-mill ZM1) having pores of 250 μm, taking care to keep the seeds cooled. To determine their α-amylase content, the milled seeds were extracted with 10 volumes of 0.5 M glycine buffer pH 9.0 with 10 mM CaCl, during 30 min at 0_C. The supernatant was used for α-amylase determination by the Phadebas method (Pharmacia Diagnostics). The units are referred to as TAU (thermostable α-amylase units).
Liquefaction tests were carried out as follows: starch slurry (composition: 3.3 kg corn or potato starch, D.S. (Dry Substance) 98% (2.904 kg starch): 5.45 l H2O: D.S. of slurry becomes 33%: the pH was corrected to 6.5 wish 1 N sulfuronic acid or 1 N NaOH. Either milled seeds or microbial α-amylase were added to an amount equivalent to 4.4 T.A.U./g D.S.) is heated to 100° C. as rapidly as possible and this temperature is maintained for 10 minutes. The slurry is then brought to 95° C. and maintained at that temperature for 2 hours. Afterwards, the samples were acidified with H2SO4 to obtain pH 3.5 and placed in a boiling water bath for 10 minutes in order to stop enzymatic activity before the DE (dextrose equivalents) and hydrolysis pattern were determined by HPLC. A column of BIORAD HPX-42A was used for HPLC analysis with demineralized water as eluent.
The oligosaccharide pattern obtained from the hydrolysis of potato and corn starch using A) transformed plant seeds; B) Maxamyl• (Bacillus licheniformis α-amylase obtained from Gist-brocades N. V., Delft, The Netherlands): and C) Dexlo•CL (Bacillus amyloliquefaciens α-amylase from Gist-brocades) were compared (
The binary vector pMOG1068 [Cru-A (promoter)-AlMV (leader)-PDI (signal peptide)-PDI (coding sequence +KDEL retention signal)-Cru-A (terminator)-RB] consists of the PDI coding region (and the PDI signal peptide and the KDEL ER retention signal) under control of the Cruciferin A (Cru A) promoter and terminator sequences. Ar. AlMV leader sequence is included. The backbone of the expression construct is the binary vector pMOG800 (a derivative of pMOG23, in which an additional KpnI restriction site was introduced into the polylinker between EcoRI and SmaI). This plasmid contains between the left and right borders of T-DNA a kanamycin resistance gene for selection of transgenic plant cells. A sample of E. coli DH5α, harbouring pMOG800, was deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on Aug. 12, 1993 under number CBS 414.93). The binary vector was cloned as follows.
The cloning vector pUC29 was made by introducing a synthetic oligo adaptor:
into the EcoRI and SstI sites of pUC18. This introduces BglII, ClaI, and NcoI sites into the polylinker of pUC18. pUC28 was subsequently linearized with SalI and NcoI.
The Cruciferin A promoter was fused to the AMV leader sequence in a three way ligation in which the (SalI-NcoI) linearized pUC28 vector, a 101 bp SalI-PvuII fragment from the CruA promoter (see Example 2) and the oligo primers:
AMV I/AMV IT:
were used. The vector was subsequently linearized with NcoI and EcoRI.
The cDNA clone of Protein Disulfide Isomerase (PDI) was isolated from alfalfa (Shorrosh and Dixon 1992, The Plant Journal 2, 51-58) by PCR amplification. Part of the clone was amplified by use of the primers:
Hereby a novel NcoI site was introduced at 180 bp downstream of the ATG start codon. The resulting 186 bp NcoI—SspI PCR fragment was cloned together with an internal SspI—EcoRI PDI fragment (1068 bp) into the pUC28 vector already containing the CruA—AlMV leader fusion (and which was linearized with NcoI and EcoRI).
The 5′ part of the PDI gene was amplified with the primers:
By using these primers a novel NcoI site was created at the ATG start codon of PDI and a novel NcoI site was created at 180 bp downstream of the ATG codon. At the same time an internal EcoRI site at 166 bp downstream of the ATG codon was destroyed. The mutations are all silent.
The 3′ part of PDI coding region was amplified with the primers:
This creates a BglII site downstream of the stop codon. The resulting 112 bp EcoRI—BglII PCR fragment was used in a three way ligation with the CruA terminator (a 219 bp BglII-XhoI fragment) into pMOG800 linearized with EcoRI and XhoI. The resulting plasmid was linearized with EcoRI and used to clone in the CruA promoter as a 2574 bp EcoRI-SalI fragment and the CruA AMV 5′PDI fusion as a 1586 bp SalI-EcoRI fragment. The resulting bianary vector was designated pMOG1068. The binary vector was finally transferred to Agrobacterium tumefaciens strain MOG 301 (Hood et al. 1993, Transgenic Res. 2:208-213).
Transgenic Brassica napus plants were generated by Agrobacterium mediated transformation as previously described (Bade and Damm, 1995, Agrobacterium-mediated transformation of Rapeseed (Brassica napus) In: I. Potrykus and G. Spangenberg (eds.) Gene Transfer to Plants, Springer verlag Berlin).
S1-seed batches of 12 independent transgenic lines harbouring pMOG1068 were analysed for the expression of PDI by western analysis. A cDNA clone encoding the alfalfa PDI was expressed in E.coli cells and a polyclonal antiserum was raised against the expressed protein in rabbits (Shorrosh et al., 1993, Plant Physiol. 103: 719-726).
Transgenic seeds were homogenised in 50 mM sodium phosphate buffer, pH 7.5, containing 10 mM EDTA, 50 mM NaCl and 0.1% Triton X-100. The extracts were clarified by centrifugation for 5-10 minutes in an Eppendorf centrifuge. Five μg protein was loaded on a 12.5% SDS-PAA gel. The level of PDI was estimated after Western blotting by a comparison with a series of known quantities of purified PDI in a wild type canola protein background. The expression level varied between 0 and approximately 0.1% (based on total extractable protein).
The binary vector pMOG1011 (β-phaseolin (promoter)-AlMV (leader)-LOX-3 (coding sequence)-Cru-A (terminator)-RB) consists of the LOX-3 coding region under control of the β-phaseolin promoter and the Cruciferin A terminator sequences. The AlMV leader is also included in the construct. The backbone of the expression construct is the binary vector pMOG800 harbouring the NPTII gene under control of the nos promoter and multiple cloning sites between the left and right T-DNA borders. The binary vector was cloned as follows.
The β-Phaseolin promoter was isolated by PCR starting of with genomic DNA isolated from Phaseolus vulgaris cv Helda as a template and the primers:
The primers were based on the sequence of the β-Phaseolin seed storage protein gene (Gen-Bank accession number J01263, M13758). Herewith a NcoI site was created at the ATG start codon of the β-Phaseolin coding region and an EcoRI site at the 5′ end of the promoter. From the resulting PCR fragment a 906 bp EcoRI-ScaI fragment was isolated. This fragment was linked by a three way ligation with a 53 bp PvuII-NcoI fragment isolated from the CruA-AlMV fusion (Example 6) into the cloning vector pUC28 (Example 6) which was linearized with EcoRI and NcoI.
To make the exact fusion of the β-Phaseolin promoter. AlMV leader and the LOX3 cDNA two synthetic adaptors were made (Lox 21 and Lox 22):
The β-Phaseolin promoter—AlMV leader fusion was digested with EcoRI and NcoI and the resulting 959 bp fragment was used in a three way ligation with the synthetic adaptors Lox 21/Lox 22 to clone into puC18 linearized with EcoRI and KpnI.
pMOG426 (Example 2) was digested with HindIII and a synthetic adaptor:
was cloned into this HindIII site creating a XhoI site downstream of the original HindIII site of the CruA terminator resulting in plasmid pMOG426*XhoI.
A cDNA clone of Lipoxygenase-3 (LOX-3) was isolated from soybean (Yenofsky et al., 1988, Isolation and characterisation of a soybean (Glycine max) lipoxygenase-3 gene. Mol. Gen. Genet 211: 215-222). To fuse the 3′ part of the LOX3 cDNA to the CruA terminator two synthetic oligo primers were synthesized:
This adaptor destroys the 3′ internal EcoRI site of LOX3 and creates a BglII site downstream of the stop codon.
The LOX3 cDNA was digested with EcoRI and the resulting 2.2 Kb fragment was used in a three way ligation with the oligo adaptors Lox 2/Lox 3 and cloned into pMOG426*XhoI which was linearized with EcoRI and BglII. From the resulting vector a EcoRI-XhoI fragment (2468 bp) was cloned into pMOG800 linearized with EcoRI and XhoI. The resulting plasmid was used as a EcoRI digested vector to clone in the 1003 bp EcoRI-KpnI fragment (containing the fusion of β-Phaseolin, AlMV leader, and 5′LOX-3) and the 289 bp KpnI-EcoRI isolated from the LOX3 cDNA clone. The resulting plasmid was designated pMOG1011.
The binary vector was finally transferred to Agrobacterium tumefaciens strain MOG 101 (Hood et al. 1993, Transgenic Res. 2: 208-218).
Transgenic Arabidopsis thaliana cv. C24 plants were generated by Agrobacterium mediated transformation as described (Valvekens et al., 1928, Proc. Natl. Acad. Sci. 85: 5536-5540).
S1-seed batches of 18 primary transformants transgenic for pMOG1219 were analysed for the expression of LOX-3 by Western analysis. Polyclonal antibodies against pea lipoxygenases 2 and 3 were raised in rabbits and obtained from Carlsberg laboratories (Copenhagen, DK). A sample of the antiserum was obtained. The antiserum crossreacts with soybean LOX-1 and LOX-3 but not with soybean LOX-2.
Transgenic seeds were homogenised in 50 mM sodium phosphate buffer, pH 7.5, containing 10 mM EDTA, 50 mM NaCl and 0.1% Triton X-100. The extracts were clarified by centrifugation for 5-10 minutes in an Eppendorf centrifuge. Twenty μg protein was loaded on a 12.5% SDS-PAA gel. The level of LOX-3 in the transgenic seed batches was compared to the expression level of the protein in a soybean mutant lacking LOX-1 but expressing both LOX-2 and LOX-3 at wild type levels (Wang et al. 1994, Proc. Natl. Acad. Sci. 91: 5828-5832) The expression level was approximately 100-200 times lower than in the mutant soybean.
A cDNA clone encoding cyclodextrin glucano-transferase (CGTase) from Bacillus circulans strain 251 (Lawson et al. 1994, J. Mol. Biol. 236, 590-600) was obtained from Dr. L. Dijkhuizen (State University Groningen). A binary expression construct, pMOG1160, (Cru-A (promoter)-Ω+4 (leader)-PR-S (signal peptide)-CGTase (coding sequence)-Cru-A (terminator)] was generated. The backbone of the binary expression construct is the binary vector pMOG800 harbouring between the left and right T-DNA borders the NPTII gene under control of the Nos promoter and multiple cloning sizes.
The cloning of pMOG1160 took place as follows:
A 101 bp SalI-PvuII fragment of the CruA promoter (Example 2 this patent) was isolated and cloned with oligo primers (Cru-Omega I/Cru-Omega II), containing the complete Omega+4 leader (Downson Day et al., Plant Mol. Biol. (1993) 23, 97-109) and part of the CruA promoter into the cloning vector pUC28 (see above) linearized with SalI and NcoI.
From this fusion a 86 bp SalI-NcoI fragment was isolated and cloned with two oligo primers (Trans T/Trans II) containing the complete PR-S signal sequence:
into pUC18 linearized with SalI-SphI, this vector was designated pTCV210.
A cDNA clone encoding cyclodextrin glucano-transferase (CGTase) from Bacillus circulans strain 251 (Lawson et al. 1994, Nucleotide sequence and X-Ray structure of Cyclodextrin Glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J. Mol. Biol. 236, 590-600) was obtained from Dr. L. Dijkhuizen (State University Groningen). This cDNA clone was used as a template in a PCR reaction with the primers:
These primers will create a SphI site just upstream of the mature CGTase gene and a BglII site directly downstream of the stop codon. The resulting 2082 bp PCR product was digested with SphI and BglII and cloned by a three way ligation together with the previously described CruA- Omega+4-PRS signal fragment (246 bp) digested with SalI-SphI into the cloning vector pUC28 linearized with SalI-BglII. The resulting 5plasmid was digested with SalI and BglII and the 2328 bp fragment was isolated and ligated with a 218 bp BglII-HindIII fragment isolated from pMOG426 (see above) containing the CruA terminator into pUC18 linearized with SalI-HindIII. This vector was used to isolate the 2546 bp SalI-HindIII fragment and cloned together with the 5′ part of the CruA promoter as a EcoRI-SalI fragment into pMOG800 linearized with EcoRI and HindIII resulting in pMOG1160.
The binary vector was finally transferred to Agrobacterium tumefaciens strain MOG 101 (Hood et al. 1993, Transgenic Res. 2: 208-218).
Transgenic Arabidopsis thaliana cv. C24 plants were generated by Agrobacterium mediated transformation as described (Valvekens et al., 1988, Proc. Natl. Acad. Sci. 65, 5536-5540).
S1 seed batches of primary Arabidopsis transformants transgenic for pMOG1160 were analysed for the expression of CGTase by Western analysis. As a standard, purified CGTase (Penniga et al. 1995, Biochemistry 34: 3368-3376) in a wild type protein background was included. The (polyclonal) antiserum that was used for the analysis was raised in rabbits against purified protein and was obtained from Lubbert Dijkhuizen, State University Groningen.
Transgenic seeds were homogenised in 50 mM sodium phosphate buffer, pH 7.5, containing 10 mM EDTA, 50 mM NaCl and 0.1% Triton X-100. The extracts were clarified by centrifugation for 5-10 minutes in an Eppendorf centrifuge. Ten μg protein was loaded on a 12.5% SDS-PAA gel. The level of CGT-ase expression varied between 0 and 2-3% based on total soluble protein.
A binary expression construct, pMOG1346 ,glutelin (promoter)-Actin1 (leader+intron)-synthetic XlnA (coding region)-glucelin (terminator)] was generated harbouring the synthetic XlnA coding region under control of the rice glutelin promoter and rice glutelin terminator sequences. The actin leader and glutelin signal peptide were included in the construct as well. The backbone of this expression construct is the binary vector pMOG1006 harbouring between the left and right T-DNA borders the HPTII gene under control of the 355 CaMV promoter and multiple cloning sites.
A cDNA clone encoding the endoxylanase from Aspergillus niger (XlnA) has been described in the literature (de Graaff et al., 1994, Mol. Microbiol. 12: 479-490).
Based on the sequence of this cDNA clone and the sequence of the rice glutelin signal peptide a synthetic Gt1-XlnA gene fusion was constructed. The codon usage of the synthetic gene was adapted to what is expected to be optimal for translation effieciency in monocotelydenous plants. At the ATG of the synthetic Gt1-XlnA gene a NcoI site was created and 3′ of the TGA stop codon a BamHI site was introduced. The synthetic gene was subcloned into the cloning vector pUC28 (see above) linearized with BamHI and NcoI and the correct sequence was confirmed by sequencing of the entire gene.
The Glutelin 1 terminator was isolated by PCR using genomic DNA isolated from Oryza Sativa cv Taipei 309 as a template and the primers:
The resulting 480 bp PCR fragment was digested with XbaI and BglII and cloned into the pUC28 Gt1-XlnA vector linearized with BamHI-XbaI, fusing the synthetic Gt1-XlnA to the Gt1 terminator.
The Actin1 leader and intron were isolated by PCR using the pAct1-D plasmid obtained from R. Wu (Zhang W. et al., Analysis of Rice Actl 5′ region activity in transgenic rice plants. The Plant Cell (1991) 3: 1155-1165) as a template and the primers (Bak-Act-08/PZT-Act-06):
From this PCR fragment a 551 bp EcoRV-NcoI fragment was isolated.
The 3′part of the Rice Glutilin promoter was isolated by PCR using the pGTl plasmid obtain from N. Murai (ref.: Zheng Z. et al., 5′ distal and proximal cis-acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene. The Plant Journal (1993), 4: 357-366) as a template and the primers:
From this PCR fragment a 522 bp EcoRV-ClaI fragment was isolated. This fragment and the previous EcoRV-NcoI actin leader fragment were cloned into pUC28 (see above) digested with NcoI-ClaI. From this plasmid a 1073 bp NcoI-ClaI fragment was isolated and cloned by a three way ligation with the 1107 bp XbaI-NcoI fragment containing the fusion of the synthetic Gt1-XlnA and Glutelin terminator into pUC28 linearized with XbaI and ClaI.
The remaining part of the rice glutelin promoter (approximately 4.5 Kb) was isolated from plasmid pTRA311 also obtained from N. Murai as a KpnI-ClaI fragment. This fragment and a XbaI-ClaI fragment from the previous plasmid were cloned into pMOG1006 linearized with XbaI and KpnI.
The binary vector was finally transferred to Agrobacterium tumefaciens strain EHA 105 (Hood et al. 1993, Transgenic Res. 2: 209-218).
Transgenic rice plants were generated by Agrobacterium mediated transformation as described (Hiei et al. (1994), The Plant Journal 6: 271-282).
Seed batches (S1) of primary rice transformants transgenic for pMOG1327 were analysed for the expression of xylanase by using an activity assay as described (Maat, J. et al., 1992, Xylanases and their application in bakery In: Xylans and Xylanases, J. Visser ed., Elsevier Science Publishers B. V.). Seeds derived from transgenic plants showed an enhanced level of xylanase activity compared to the non-transgenic control samples.
While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without parting from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, plant, seed, process, process step or steps to the object, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto.
Pup-loaves were baked from 150 g dough pieces obtained by mixing 200 g wheat flour (100%), 106 ml water (53%), 1.2 g instant dry baker's yeast (0.6%: Gist-brocades N. V., Delft, The Netherlands), 4 g NaCl (2%), 400 mg CaCl2.2H2) (0.2%), 10 mg fungal α-amylase P100 (Gist-brocades, 2250 SKB/kg flour) and a variable number of units of endo-xylanase (xyl A) activity. After mixing for 6 minutes and 15 seconds at 52 r.p.m. in a pin mixer, the dough was divided, proofed for 45 minutes at 31° C., punched, proofed for an additional 25 minutes, molded and panned. After a final proof of 70 minutes at 31° C. the dough was baked for 20 minutes in an oven at 250° C. Loaf volume was determined by the rapeseed displacement method. The results are summarized in the Table below.
*= score from 1 (lowest quality) to 10 (highest quality)
| Number | Date | Country | Kind |
|---|---|---|---|
| 91200688.9 | Mar 1991 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09149310 | Feb 1998 | US |
| Child | 11214413 | Aug 2005 | US |
| Parent | 08146422 | Nov 1993 | US |
| Child | 08626554 | Apr 1996 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08626554 | Apr 1996 | US |
| Child | 09149310 | Feb 1998 | US |
| Parent | 07756994 | Sep 1991 | US |
| Child | 08146422 | Nov 1993 | US |
| Parent | 07498561 | Mar 1990 | US |
| Child | 07756994 | Sep 1991 | US |
