Patent Grant
8252555
This application is a 371 National Phase entry of PCT/EP2007/064471, filed 21 Dec. 2007, and claims the benefit of European Patent Application Serial No. 06127363.7, filed 29 Dec. 2006.
The present invention relates to nucleotide sequences encoding enzymatically active cobalamin-methionine synthase and functional fragments thereof being modified in comparison to the respective wild-type enzyme such that said enzymes show a reduced product inhibition by methionine. The present invention also relates to polypeptides being encoded by such nucleotide sequences and host cells comprising such nucleotide sequences. Furthermore, the present invention relates to methods for producing methionine in host organisms by making use of such nucleotide and amino acid sequences.
Currently, worldwide annual production of methionine is about 500,000 tons. Methionine is the first limiting amino acid in livestock of poultry feed and due to this, mainly applied as feed supplement. In contrast to other industrial amino acids, methionine is almost exclusively applied as a racemate produced by chemical synthesis. Since animals can metabolise both stereoisomers of methionine, direct feed of the chemically produced racemic mixture is possible (D'Mello and Lewis, Effect of Nutrition Deficiencies in Animals: Amino Acids, Rechgigl (Ed.), CRC Handbook Series in Nutrition and Food, 441-490, 1978).
However, there is still a great interest in replacing the existing chemical production by a biotechnological process. This is due to the fact that at lower levels of supplementation L-methionine is a better source of sulfur amino acids than D-methionine (Katz et al., (1975) Poult. Sci., 545: 1667-74). Moreover, the chemical process uses rather hazardous chemicals and produces substantial waste streams. All these disadvantages of chemical production could be avoided by an efficient biotechnological process.
For other amino acids such as glutamate, fermentation production methods are known. For these purposes, certain microorganisms such as Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum) have proven to be particularly suited. The production of amino acids by fermentation also has the particular advantage that only L-amino acids are produced. Further, environmentally problematic chemicals such as solvents, etc. which are used in chemical synthesis are avoided. However, fermentative production of methionine by microorganisms will only be an alternative to chemical synthesis if it allows for the production of methionine on a commercial scale at a price comparable to that of chemical production.
Hence, the production of L-methionine through large-scale culture of bacteria developed to produce and secrete large quantities of this molecule is a desirable goal. Improvements to the process can relate to fermentation parameters, such as stirring and supply of oxygen, or the composition of the nutrient media, such as the sugar concentration during fermentation, or the working up of the product by, for instance, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis and mutant selection are also used to improve the output properties of these methionine-producing microorganisms. High production strains which are resistant to antimetabolites or which are auxotrophic for metabolites of regulatory importance are obtained in this manner.
Recombinant DNA technology has also been employed for some years for improving microorganism strains which produce L-amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
Rückert et al. (Journal of Biotechnology (2003), 104: 213-228) provide an analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum. Known functions of MetZ (also known as MetY) and MetB could be confirmed and MetC (also known as AecD) was proven to be a cystathionine-β-lyase. Further, MetE and MetH, which catalyse the conversion of L-homocysteine to L-methionine, were identified in this study.
WO 02/097096 discloses nucleotide sequences from coryneform bacteria which code for the McbR repressor gene (also known as MetD) and processes for the preparation of amino acids using bacteria in which this McbR repressor gene is attenuated. According to WO 02/097096, the attenuation of the transcriptional regulator McbR improves the production of L-methionine in coryneform bacteria. It is further described in WO 02/097096 that, in addition to the attenuation of the McbR repressor gene, enhancing or overexpressing the MetB gene which codes for cystathionine-γ-synthase is preferred for the preparation of L-methionine.
Selection of strains improved for the production of a particular molecule is a time-consuming and difficult process. Therefore, there is still a great need for microorganisms which efficiently produce L-methionine and/or have significantly increased contents of L-methionine which can be utilized for obtaining methionine.
It is an object of the present invention to provide nucleotide sequences which encode enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof having the property of reduced product inhibition by methionine. Such nucleotide sequences encode preferably enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in their amino acid sequence compared to the respective wild-type amino acid sequence such that these enzymes show reduced product inhibition by methionine, i.e. the enzymatic activity is inhibited by methionine to a lesser extent than for the wild-type enzymes and polypeptides. Such nucleotide sequences may e.g. be DNA and/or RNA sequences with DNA sequences being preferred.
It is a further object of the present invention to provide polypeptides and preferably proteins which are encoded by such nucleotide sequences.
Yet another object of the present invention is to provide vectors which comprise such nucleotide sequences and can be used for expression of such nucleotide sequences and polypeptides in host cells.
Another object of the present invention relates to host cells which express the aforementioned nucleotide and polypeptide sequences.
Yet another object of the present invention relates to the use of such nucleotide and polypeptide sequences for producing methionine and/or increasing the efficiency of methionine production in host organisms.
The present invention also relates to methods for producing methionine by expressing said nucleotide and polypeptide sequences in host organisms.
According to one embodiment of the invention, nucleotide and preferably DNA sequences are provided which encode an enzymatically active cobalamin-dependent methionine synthase or functional fragments thereof having the property of reduced product inhibition by methionine. In one embodiment such nucleotide and preferably DNA sequences encode enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in their amino acid sequence compared to the respective wild-type amino acid sequences, such that the enzymatic activity of said enzymatically active cobalamin-dependent methionine synthases or of said functional fragments shows reduced product inhibition as a consequence of the at least one mutation. This means that the enzymatic activity of the mutated enzymes or functional fragments thereof is inhibited by methionine to a lesser extent compared to the respective wild-type sequences.
Such nucleotide and preferably DNA sequences may also allow the construction of host organisms which product and secrete preferably large quantities of the desired molecule, i.e. L-methionine.
In a further embodiment of the present invention, such nucleotide and preferably DNA sequences encode enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in SEQ ID NO: 1 such that the encoded polypeptides show reduced product inhibition by methionine compared to the respective wild-type polypeptides.
Yet another embodiment of the present invention relates to nucleotide and preferably DNA sequences that encode enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in their homocysteine-binding domain such that these polypeptides show reduced product inhibition by methionine. A typical homocysteine-binding domain is that of MetH of C. glutamicum. The corresponding DNA sequence is SEQ ID NO: 24 while the amino acid sequence is SEQ ID NO: 2.
In yet another embodiment of the present invention, nucleotide and preferably DNA sequences encode enzymatically active cobalamin-dependent methione synthases or functional fragments thereof which carry at least one mutation in SEQ ID NOs: 3 to 18 such that these polyp eptides show reduced product inhibition by methionine.
One embodiment of the present invention relates to nucleotide and preferably DNA sequences which encode enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in a position corresponding to M33, F86 or S134 of the cobalamin-dependent methionine synthase MetH of C. glutamicum. The DNA sequence of MetH of C. glutamicum is that of SEQ ID NO: 23. The amino acid sequence of MetH of C. glutamicum is that of SEQ ID NO: 1.
The present invention in one embodiment also relates to nucleotide and preferably DNA sequences which encode polypeptides that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the polypeptides being encoded by the afore-mentioned nucleotide sequences which carry at least one mutation in these sequences compared to the wild-type sequences such that the resulting enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof show reduced product inhibition by methionine.
Thus, the present invention on one embodiment relates to nucleotide and preferably DNA sequences which encode polypeptides that are at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the polypeptides being encoded by any of SEQ ID NOs: 1 to 18 with the proviso that these polypeptides carry at least one mutation in these sequences such that the resulting enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof show reduced product inhibition by methionine.
Yet another embodiment of the present invention relates to nucleotide and preferably DNA sequences that are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the aforementioned nucleotide and preferably DNA sequences.
Thus, in one embodiment the present invention relates to nucleotide and preferably DNA sequences that are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to e.g. SEQ ID NO: 23 or SEQ ID NO: 24 with the proviso that the nucleotide sequences carry additionally at least one mutation such that the resulting enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof show reduced product inhibition by methionine.
In preferred embodiments these nucleotide and preferably DNA sequences are isolated or recombinant nucleotide and preferably DNA sequences.
Another embodiment of the present invention relates to nucleotide and preferably DNA sequences that hybridise under stringent conditions to the aforementioned nucleotide sequences.
Other embodiments of the present invention relate to nucleotide and preferably DNA sequences encoding enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation with respect to the corresponding wild type sequences, the enzymatic activity of which is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and preferably at least a factor of 2, 3, 4, 5, 10, 20, 50, 100, 200, 500 or 1000 less inhibited in the presence of 20 mM methionine compared to respective wild-type cobalamin-dependent methionine synthases or functional fragments thereof.
The present invention also relates to vectors which comprise the aforementioned nucleotide and preferably DNA sequences in operative linkage to promoter and termination sequences such that these nucleotide sequences can be expressed in host organisms.
Other embodiments of the present invention relate to polypeptides which are encoded by the aforementioned nucleotide and preferably DNA sequences.
Yet another embodiment of the present invention relates to host cells which comprise the aforementioned nucleotide and preferably DNA sequences.
In one embodiment of the present invention, such host cells express the aforementioned nucleotide and preferably DNA sequences, preferably from one of the aforementioned vectors.
According to a further embodiment of the present invention, such host cells are selected from microorganisms and yeasts. In one embodiment, the microorganism is selected from the group consisting of e.g. coryneform bacteria, microbacteria, streptomycetaceae, salmonella, Escherichia coli, Shigella, Bacillus, Serratia, Pseudomonas, S. coel or Thermotoga maritima.
While any host cell or host organism in accordance with the present invention must comprise the above mentioned nucleotide and preferably DNA sequences which encode for enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof having reduced product inhibition by menthionine, one embodiment of the present invention relates to host cells in which additionally the endogenous gene(s) for cobalamin-dependent methionine synthase(s) is/are deleted or functionally disrupted.
Other embodiments of the invention relate to host cells and host organisms in which at least one of the nucleotide and preferably DNA sequences in accordance with the present invention is expressed and in which the amount and/or activity of at least one polypeptide being encoded by the following nucleotide sequences is increased in comparison to the corresponding initial host organism:
Another embodiment of the present invention relates to host cells and host organisms in which one of the nucleotide and preferably DNA sequences in accordance with the present invention is expressed and in which additionally the amount and/or activity of at least one polypeptide being encoded by the following nucleotide sequences is decreased with respect to the corresponding initial organism:
Another aspect of the invention relates to host cells and organisms in which the efficiency and/or yield and/or amount of methionine production is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and preferably at least by a factor of 2, 3, 4, 5, 10, 20, 50, 100 or 1000 in comparison to a host cell or host organism in which no nucleotide sequence in accordance with the invention is expressed.
Other embodiments of the present invention relate to methods for producing methionine in a host cell or organism wherein a nucleotide and preferably DNA or polypeptide sequence in accordance with the invention is expressed in the host cell. Other embodiments of the present invention relate to methods for producing methionine wherein one of the aforementioned host cells is used.
One aspect of the present invention relates to a method for producing methionine in which one of the aforementioned host cells is cultivated and methionine is subsequently isolated. The present invention also relates to the use of the aforementioned host cells for producing methionine and to the use of nucleotide and preferably DNA sequences in accordance with the present invention to produce methionine and host cells which are useful in producing methionine.
Before describing in detail exemplary embodiments of the present invention, the following definitions are given.
The terms “nucleotide sequences in accordance with the present invention” and “DNA sequences in accordance with the present invention” refer to the corresponding sequences as mentioned above that encode polypeptides being enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof that show a reduced product inhibition by methionine. The term “reduced product inhibition by methionine” will be defined further below. The term “polypeptide” means to encompass proteins and typically relates to polypeptides with more than 20 amino acids.
The term “efficiency of methionine synthesis” describes the carbon yield of methionine. This efficiency is calculated as a percentage of the energy input which enters the system in the form of a carbon substrate. Throughout the invention this value is given in percent values ((mol methionine) (mol carbon substrate)−1·100), unless indicated otherwise. Preferred carbon sources according to the present invention are sugars, such as mono-, di-, or polysaccharides. For example, sugars selected from the group consisting of glucose, fructose, manose, galactose, libose, sorbose, lactose, maltose, sucrose, raffinose, starch or cellulose may serve as particularly preferred carbon sources.
The term “increased efficiency of methionine synthesis” relates to the comparison between an organism being a host cell that has been genetically modified to express nucleotide and preferably DNA sequences in accordance with the present invention and which has a higher efficiency of methionine synthesis compared to the initial organism which does not express the nucleotide and preferably the DNA sequences in accordance with the present invention.
The initial organism which does not express nucleotide and preferably DNA sequences in accordance with the present invention may be a wild-type organism. Alternatively, it may be an organism that has already been optimised for methionine production and thus over-expresses certain genes of the methionine synthesis pathway. Alternatively, an initial organism which has already been optimised for methionine production may show a reduced expression for certain enzymes of the methionine pathway.
The terms “methionine pathway” and “methionine biosynthesis pathway” are art-recognised and describe a series of reactions which take place in a wild-type organism and lead to the biosynthesis of L-methionine. These pathways may vary from organism to organism. The details of an organism-specific pathway can be taken from textbooks and the scientific literature on the interne website HyperTextTransferProtocol://WorldWideWeb.genome.jp/kegg/metabolism.HyperTextMarkupLanguage, wherein “HyperTextTransferProtocol”=“http”, “WorldWideWeb”=“www”, and “HyperTextMarkupLanguage”=“html”. In particular, a methionine pathway within the meaning of the present invention is shown in
The term “yield of methionine” describes the yield of methionine which is calculated as the amount of methionine obtained per weight cell mass.
The terms “organism”, “host organism”, “host cell” or “microorganism” for the purposes of the present invention refer to any organism that is commonly used for the production of amino acids such as methionine. In particular, these terms relate to procaryots, lower eucaryots and fungi. A preferred group of the above-mentioned organisms comprises actino bacteria, cyano bacteria, proteo bacteria, Chloroflexus aurantiacus, Pirellula sp. 1, halo bacteria and/or methanococci, preferably coryneform bacteria, myco bacteria, streptomyces, salmonella, Escherichia coli, Shigella, Pseudomonas, S. coel or Thermotoga maritima.
Particularly preferred microorganisms are selected from Corynebacterium glutamicum, Escherichia coli, microorganisms of the genus Bacillus, particularly Bacillus subtilis, microorganisms of the genus Streptomyces, or of the genus Thermotoga, particularly Thermotoga maritima.
The organisms of the present invention may, however, also comprise yeasts such as Schizosaccharomyces pombe or S. cerevisiae and Pichia pastoris.
The terms “L-methionine over-producing organism”, “methionine over-producing organism” or “methionine-producing organism” for the purposes of the present invention refer to an “organism”, “host organism” or “microorganism” in which compared to an initial organism which does not express nucleotide and preferably DNA sequences in accordance with the present invention, the efficiency and/or yield and/or amount of methionine production is increased at least by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or at least by a factor of 2, 3, 4, 5, 10, 15, 20, 50, 100, 500 and 1000 or more.
The term “metabolite” refers to chemical compounds that are used in the metabolic pathways of organisms as precursors, intermediates and/or end products. Such metabolites may not only serve as chemical building units, but may also exert a regulatory activity on enzymes and their catalytic activity. It is known from the literature that such metabolites may inhibit or stimulate the activity of enzymes (Stryer, Biochemistry, (1995) W.H. Freeman & Company, New York, N.Y.).
For the purposes of the present invention, the term “external metabolite” comprises substrates such as glucose, sulfate, thiosulfate, sulfite, sulfide, ammonia, oxygen etc.
If, in the context of the present invention, reference is made to the content of a nucleotide sequence or the content of a polypeptide encoded by the nucleotide sequence, this refers to the amount of nucleic acid and polypeptide being encoded by such nucleic sequences as they can be determined for the respective host organism comprising such nucleotide sequences or polypeptides.
If reference is made to the activity of a nucleotide sequence this typically, for the purposes of the present invention, means to encompass the activity of the polypeptide or protein that is encoded by such a nucleotide sequence.
If, in the context of the present invention, it is stated that the amount of nucleotide sequence is increased with respect to a wild-type or initial organism, this means that the amount of this nucleotide sequence and the amount of the polypeptide that is encoded by the nucleic acid are increased in comparison to an organism which is not genetically manipulated with respect to this specific nucleotide sequence or polypeptide. This may be achieved by introducing a corresponding exogenous nucleotide sequence into a host organism and the comparison then refers to the host organism expressing the nucleotide sequence and the initial organism into which the nucleotide sequence has not been introduced. Alternatively, the amount of nucleotide sequence may be increased by manipulating other regulatory sequences or the endogenous sequences within an organism.
Thus, increasing the amount of a nucleotide sequence may be achieved by introducing exogenous sequences or manipulating endogenous sequences that are responsible for the level of expression of the respective nucleotide sequences.
If, in the context of the present invention, it is stated that the activity of a nucleotide sequence is increased with respect to an initial organism, this refers to a situation where typically the activity of the polypeptide that is encoded by this nucleotide sequence is increased in comparison to the initial organism. Increasing the activity may be achieved by increasing the amount of the nucleotide sequence, and/or by introducing mutations in nucleotide sequences encoding polypeptides with increased activity.
Thus, increasing the activity of a nucleotide sequence and the polypeptide being encoded thereby may be achieved by either introducing exogenous nucleotide sequences and/or introducing mutations into the regulatory and coding sequences of endogenous sequences that are responsible for expressing the sequence of interest.
If, in the context of the present invention, it is stated that the content (amount) and/or activity of a nucleotide sequence, and consequently of the polypeptide being encoded thereby, is decreased in comparison to an initial organism, the above definitions are to be applied mutatis mutandis.
The terms “express,” “expressing,” “expressed” and “expression” refer to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) or a nucleotide sequence. The expression can be done by genetic alteration of the e.g. microorganism that is used as an initial starting organism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product such as a polypeptide at an increased level relative to that produced by the initial microorganism or in a comparable microorganism which has not been altered. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).
The terms “overexpress”, “overexpressing”, “overexpressed” and “overexpression” refer to expression of a gene product (e.g. a methionine biosynthetic enzyme or a gene or a pathway or a reaction defined and described in this application) or a nucleotide sequence at a level greater than that present prior to a genetic alteration of the initial microorganism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product or nucleotide sequence at an increased level relative to that produced by the initial microorganism. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). Examples for the overexpression of genes in organisms such as C. glutamicum can be found in Eikmanns et al (Gene. (1991) 102, 93-8).
In some embodiments, a microorganism can be physically or environmentally altered to express a gene product or nucleotide sequence at an increased or lower level relative to level of expression of the gene product or nucleotide sequence by the initial microorganism. For example, a microorganism can be treated with or cultured in the presence of an agent known or suspected to increase transcription of a particular nucleotide sequence and/or translation of a particular nucleotide sequence such that transcription and/or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular nucleotide sequence or gene and/or translation of a particular nucleotide sequence or gene product such that transcription and/or translation are enhanced or increased.
The terms “disrupt”, “disrupted”, “disruption”, “deregulate,” “deregulated” and “deregulation” refer to alteration or modification of at least one gene or nucleotide sequence in e.g. a microorganism, wherein the alteration or modification results in increasing efficiency or yield of methionine production in the microorganism relative to methionine production in absence of the alteration or modification. In some embodiments, a gene or nucleotide sequence that is altered or modified encodes an enzyme in a biosynthetic pathway, such that the level or activity of the biosynthetic enzyme in the microorganism is altered or modified. In some embodiments, at least one gene that encodes an enzyme in a biosynthetic pathway is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene. In some embodiments, the biosynthetic pathway is the methionine biosynthetic pathway. Deregulation also includes altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity. Also, deregulation further encompasses genetic alteration of genes encoding transcriptional factors (e.g., activators, repressors) which regulate expression of genes in the methionine and/or cysteine biosynthetic pathway.
The phrase “deregulated pathway or reaction” refers to a biosynthetic pathway or reaction in which at least one gene that encodes an enzyme in a biosynthetic pathway or reaction is altered or modified such that the level or activity of at least one biosynthetic enzyme is altered or modified. The phrase “deregulated pathway” includes a biosynthetic pathway in which more than one gene has been altered or modified, thereby altering level and/or activity of the corresponding gene products/enzymes. In some cases the ability to “deregulate” a pathway (e.g., to simultaneously deregulate more than one gene in a given biosynthetic pathway) in a microorganism arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed an “operon.” In other cases, in order to deregulate a pathway, a number of genes must be deregulated in a series of sequential engineering steps.
The term “operon” refers to a coordinated unit of genetic material that contains a promoter and possibly a regulatory element associated with one or more, preferably at least two, structural genes (e.g., genes encoding enzymes, for example, biosynthetic enzymes). Expression of the structural genes can be coordinately regulated, for example, by regulatory proteins binding to the regulatory element or by anti-termination of transcription. The structural genes can be transcribed to give a single mRNA that encodes all of the structural proteins. Due to the coordinated regulation of genes included in an operon, alteration or modification of the single promoter and/or regulatory element can result in alteration or modification of each gene product encoded by the operon. Alteration or modification of a regulatory element includes, but is not limited to, removing endogenous promoter and/or regulatory element(s), adding strong promoters, inducible promoters or multiple promoters or removing regulatory sequences such that expression of gene products is modified, modifying the chromosomal location of the operon, altering nucleic acid sequences adjacent to the operon or within the operon such as a ribosome binding site, codon usage, increasing copy number of the operon, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of the operon and/or translation of the gene products of the operon, or any other conventional means of deregulating expression of genes routine in the art (including, but not limited to, use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).
In some embodiments, recombinant microorganisms described herein have been genetically engineered to overexpress a bacterially derived gene or gene product. The terms “bacterially-derived” and “derived-from bacteria” refer to a gene which is naturally found in bacteria or a gene product which is encoded by a bacterial gene.
Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in organisms. The term “amino acid” is well known in the art. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the non-proteinogenic amino acids are not normally found in proteins (see Ullmann's Encyclopaedia of Industrial Chemistry, Vol. A2, pages 57-97, VCH, Weinheim (1985)). Amino acids may be in the D- or L-optical configuration, although L-amino acids are generally the only type found in naturally-occurring proteins. Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pages 578-590 (1988)).
The essential amino acids, i.e. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, which are generally a nutritional requirement due to the complexity of their biosynthesis, are readily converted by simple biosynthetic pathways to the remaining 11 non-essential amino acids, i.e. alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine.
Higher animals retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for normal protein synthesis to occur. Apart from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetic, agricultural and pharmaceutical industries.
Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals, such as poultry and swine. Glutamate is most commonly used as a flavour additive, and is widely used throughout the food industry as are aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are all utilized in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetic industries. Threonine, tryptophan and D/L-methionine are common feed additives (Leuchtenberger, W. (1996), Amino acids—technical production and use, p. 466-502 in Rehm et al. (editors) Biotechnology, Vol. 6, Chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthetic of synthetic amino acids and proteins such as N-acetyl cysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan and others described in Ullmann's Encyclopaedia of Industrial Chemistry, Vol. A2, p. 57-97, VCH: Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation therefor (see Umbarger H. E. (1978), Ann. Rev. Biochem. 47:533-606). Glutamate is synthesized by the reductive amination of α-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a three-step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by transferral of the side-chain β-carbon atom to tetrahydrofolate, in a reaction catalysed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose-4-phosphate and phosphoenolpyruvate in a nine-step biosynthetic pathway that differ only at the final two steps after the synthesis of prephenate. Tryptophan is also produced from these two initial molecules, but its synthesis is an eleven-step pathway. Tyrosine may also be synthesized from phenylalanine in a reaction catalysed by phenylalanine hydroxylase. Alanine, valine and leucine are all biosynthetic products of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine and lysine are each produced by the conversion of aspartate. Isoleucine may be formed from threonine. A complex nine-step pathway results in the production of histidine from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be stored and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L., Biochemistry, 3rd edition, Chapter 21 “Amino acid degradation and the urea cycle”, p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesise them.
Amino acid biosynthesis can be regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L., Biochemistry, 3rd edition, Chapter 24: “Biosynthesis of amino acids and heme”, p. 575-600 (1988)). If this feedback inhibition is mediated by an amino acid forming the product of the regulated reaction or pathway, one typically speaks of “product inhibition”. Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell.
The Gram-positive soil bacterium Corynebacterium glutamicum is widely used for the industrial production of different amino acids. Whereas the biosynthesis of lysine and glutamate, the main industrial products, has been studied for many years, knowledge about the regulation of the methionine biosynthetic pathway is limited.
However, at least the key enzymes of the pathway are known (see
Recently, a putative transcriptional regulator protein of the TetR-family was found (Rey et al. (2003), Journal of Biotechnology, 103: 51-65). This regulator was shown to repress the transcription of several genes belonging to methionine and sulfur metabolism. The gene knockout of the regulator protein led to an increased expression of hom encoding homoserine dehydrogenase, metZ encoding O-acetylhomoserine sulfhydrolase, metK encoding S-adenosylmethionine (SAM) synthase (EC 2.5.1.6), cysK encoding cysteine synthase (EC 2.5.1.47), cysI encoding a putative NADPH dependant sulfite reductase, and finally ssuD encoding an putative alkanesulfonate monooxygenase. Rey et al. (Molecular Microbiology 2005, 56, 871-887) also found that the metB gene is significantly induced in a mcbR minus strain.
As regards the cob(I)alamin-dependent methionine synthases which, for the purposes of the present invention, are also designated as cobalamin-dependent methionine synthases or MetH, it has been shown that activity of this enzyme is inhibited by its product, i.e. methionine (Banerjee et al. (1990), Biochemistry, 29:11101-1109).
This so-called “product inhibition” of methionine probably accounts for the high need of methionine production which has been calculated to require an energy input of 7 mol ATP and 8 mol NADPH per molecule methionine (Neidhardt et al. (1990) Physiology of the Bacterial Cell: A Molecular Approach, Sunderland, Mass., USA, Sinauer Associates, Inc.). Thus, methionine is the one amino acid with respect to which a cell has to provide the most energy.
As a consequence thereof, methionine-producing organisms have evolved metabolic pathways that are under strict control with respect to the rate and amount of methionine synthesis (Neidhardt (1996) E. coli and S. typhimurium, ASM Press Washington). These regulation mechanisms include e.g. feedback control mechanisms such as the above-mentioned product inhibition of the activity of the cobalamin-dependent methionine synthase.
The product inhibition of the cobalamin-dependent methionine synthase creates a particular bottleneck when producing methionine over-producing microorganisms, as this enzyme catalyses the last step in the methionine biosynthesis pathway. Thus, microorganisms which have been optimized with respect to expression of other enzymes involved in the methionine biosynthesis pathway may ultimately prove to be unusable for efficient methionine production, because even though e.g. elevated amounts of homocysteine have accumulated in these microorganisms, homocysteine cannot be efficiently methylated to methionine, as the cells will shut off this enzymatic step once enough methionine has been produced.
As can be taken from
One of the objectives of the present invention is to resolve the limitations for the non-chemical methionine production in organisms. This and other objectives which will be become apparent from the ensuing description are solved by the independent claims. Preferred embodiments are described in the dependent claims.
The core of the present invention lies at the surprising finding that it is possible to produce mutants of cobalamin-dependent methionine synthases in which the inhibition of the enzymatic activity by the product methionine is significantly reduced.
These mutants, which show reduced product inhibition, thus continue to efficiently catalyze the methylation of homocysteine into methionine in a cobalamin-dependent manner, even when methionine levels are reached for which a microorganism will usually down-regulate the enzymatic activity of this last step. As these mutants decouple the enzymatic activity of cobalamin-dependent methionine synthase from the feedback control mechanism of product inhibition, they allow the construction of host organisms that produce methionine continuously and efficiently.
While such enzymatically active cobalamin-dependent methionine synthase mutants have been specifically isolated for the MetH enzyme of C. glutamicum, it is justified to assume that corresponding mutants exist for cobalamin-dependent methionine synthases in other organisms such as E. coli, S. coel and T. maritima. This is supported by the fact that cobalamin-dependent methionine synthases from E. coli, S. coel, C. glutamicum, Thermotoga maritima show a high degree of sequence similarity particularly in the homocysteine-binding domain which is the region that has been identified by the present invention to be most suitable for introducing mutations that reduce the product inhibition of cobalamin-dependent methionine synthases in C. glutamicum.
Before specific and preferred cobalamin-dependent methionine synthase mutants are described in more detail, an overview is given for the properties of cobalamin-dependent methionine synthases in general.
Cobalamin-dependent methionine synthase catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine generating tetrahydrofolate and methionine (Banerjee (1990) vide supra). The MetH gene from E. coli as well as from other organisms including T. maritima, S. coel and C. glutamicum have been cloned, and in some cases characterized (Banerjee (1990) vide supra; Ludwig et al. (1997) Annu. Rev. Biochem., 66:269-313; Yamada et al. (1999) Journal of Biological Chemistry, 274:33571-33576; Evans et al. (2004) Proc. Natl. Acad. Sci. USA, 101:3729-3736).
The enzyme contains a non-covalently bound cobalamin prosthetic group that functions as an intermediary in the methyl-transferase reaction. During catalysis, the enzyme shuttles between the E-methyl cobalamin and E-cob(I)alamin states, being alternately demethylated by homocysteine and remethylated by methyltetrahydrofolate.
An assay to measure the activity of methionine synthase is described in the literature (Drummond et al. (1995) Analytical Biochemistry, 228:323-329). This latter reference is specifically incorporated by reference, as far as it describes assays for the characterization of cobalamin-dependent methionine synthases. Thus, the passages starting on page 324, right column (“Materials and Methods”) to page 326, left column (“Results”) of the Drummond et al. reference form part of the disclosure of this application as far as non-radioactive assays for characterization of cobalamin-dependent methionine synthases are concerned. The assay described by Drummond et al. can be used for determining the influence of the product methionine on the enzymatic activity of cobalamin-dependent methionine synthases along the same lines as described by the aforementioned reference of Banerjee et al. (1990) vide supra for radioactive assays on page 11102, left column (“Experimental procedures”) to page 11103, left column (“Results”) and page 11103, right column (“Product inhibition data”) to page 11104, left column (“Pre-steady-state kinetic analysis of catalysis”) and Table II of the Banerjee et al. reference. As can be taken from the latter reference cobalamin-dependent methionine synthases are inhibited by methionine in a non-competitive manner.
The cobalamin-dependent methionine synthase of E. coli which is representative for other cobalamin-dependent methionine synthases from organisms such as C. glutamicum, etc. is a modular protein consisting of various domains.
The first 352 residues of the E. coli enzyme comprise a homocysteine binding region. Residues 353 to 649 are involved in the binding of methyltetrahydrofolate, while residues 650 to 896 bind the cobalamin co-factor. The carboxy terminal residues 897 to 1227 are required for reactivation of oxidized cob(II)alamin and bind adenosylsylmethyl. A 71 kDa fragment, which comprises residues 2 to 649, contains the homocysteine and methyltetrahydrofolate binding domains and catalyses methyl transfer to and from exogenous cobalamin. The 98 kDa fragment comprises both the aforementioned substrate binding regions and the cobalamin binding domain and is capable of enzymatic turnover using endogenous cobalamin co-factor.
The present invention at its core relates to enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof with reduced product inhibition by methionine.
As set out above, the present invention in one embodiment relates to enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof which carry at least one mutation in comparison to the respective wild-type sequence with the mutation having the consequence that the mutated enzymatically active cobalamin-dependent methionine synthases or fragments thereof show reduced product inhibition by methionine.
The term “functional fragment” refers to fragments of wild-type full-length versions of enzymatically active cobalamin-dependent methionine synthases which are able to catalyse the methylation of homocysteine to methionine in a cobalamin-dependent manner and which additionally carry at least one mutation in their amino acid sequence which effects reduced product inhibition by methionine.
Cobalamin-dependent methionine synthases or functional fragments thereof in accordance with the invention are thus preferably considered to show reduced impaired product inhibition if the non-competitive inhibition of the methylation of homocysteine with methyltetrahydrofolate by the enzyme is influenced to a lesser extent by methionine than for the respective wild-type cobalamin-dependent methionine synthases or functional fragments thereof.
According to the present invention, an enzymatically active cobalamin-dependent methionine synthases or a functional fragments thereof are particularly considered to show reduced product inhibition by methionine if, as a consequence of the mutation in the amino acid sequence, the inhibition of the activity by methionine, and preferably by approximately 20 mM methionine is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and preferably by at least a factor of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200, 500, 1000 or more compared to the enzymatic activity of the respective wild type enzyme or functional fragment.
If the enzymatic activity of a wild-type cobalamin-dependent methionine synthase or a functional fragment thereof is defined as 100%, cobalamin-dependent methionine synthases or functional fragments thereof with reduced product inhibition by methionine in accordance with the invention show an increased activity in the presence of methionine, and preferably of approximately 20 mM methionine of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and preferably by at least a factor of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200, 500, 1000 or more compared to the enzymatic activity of the respective wild type enzyme or functional fragment.
The influence of methionine, and preferably of approximately 20 mM methionine on the activity of either wild-type enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof and enzymatically active cobalamin-dependent methionine synthases in accordance with the present invention which have reduced product inhibition can be determined by the assay as described by Drummond et al. (vide supra).
By way of example, the cobalamin-dependent methionine synthase may be MetH of C. glutamicum and have an amino acid sequence of SEQ ID NO: 1. If a MetH version of SEQ ID NO: 1 which additionally carries at least one mutation shows a reduced influence of methionine, and preferably of approximately 20 mM methionine on the activity of this enzyme under the above test conditions, it will be considered as a cobalamin-dependent methionine synthase with reduced product inhibition in accordance with the present invention. The same would apply for a functional fragment of MetH.
Accordingly, cobalamin-dependent methionine synthases of other organisms such as E. coli, T. maritima, B. subtilis, S. coel which show a significant homology to the wild-type MetH enzyme of C. glutamicum and which additionally carry mutations that reduce the product inhibition of these enzymes in comparison to their respective wild-type enzymes under the above test conditions, will also be considered as cobalamin-dependent methionine synthases with a reduced product inhibition in accordance with the present invention.
According to the present invention, a significant sequence homology between two nucleic acid molecules or two polypeptides is generally understood to indicate that the nucleotide sequences or the amino acid sequences, respectively, of a e.g. DNA molecules or proteins are at least 30%, at least 40%, preferably at least 50%, at least 60%, at least 70%, also preferably at least 80%, particularly preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% identical. Additionally, the term “significant sequence homology” can require that the e.g. 90% identical nucleotide sequences encode polypeptides with the same function, e.g. a cobalamin-dependent methionine synthase or functional fragment thereof.
Identity of two nucleotides sequences or polypeptides is understood to be the identity of the nucleotides or amino acids over the respective entire length of the nucleotide sequences or the polypeptides respectively. Identity and Homology can be calculated using the laser gene software from DNA Star, Inc., Madison, Wis. (USA) applying the CLUSTAL method (Higgens et al. (1989), Comput. Appl. Biochi., 5(2):151). Homologies and identities for amino acid and nucleic acid sequences may also be calculated using algorithms which are based on algorithms by Niedelmann and Wunsch or Smith and Waterman. Software that may be used for these purposes are the programs Pil Aupa (J. Mol. Evolution (1987), 25, 351-360; Higgins et al. (1989) Cabgos, 5:151) or the programs Gap and Bestfit (Niedelmann und Wunsch (1970), J. Mol. Biol., 48, 443-453 and Smith and Waterman (1981) Adv. Appl. Math., 2, 482-489). For the purposes of determining the identity of two sequences, the default parameters of the above software programs are used.
An example of determining a significant sequence homolgy between cobalamin-dependent methionine synthases of different organisms is provided by the sequence alignment of
In one embodiment of the present invention, the mutations that lead to reduced product inhibition of the enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof are located in the homocysteine binding region of the proteins. For the E. coli cobalamin-dependent methionine synthase, this region has been mapped to amino acids 1 to 251.
It is well within the general knowledge of the person skilled in the art to identify corresponding domains in proteins that are related to the E. coli enzyme. In
Accordingly, one embodiment of the present invention relates to cobalamin-dependent methionine synthases which carry mutations in a sequence that is significantly homologous to SEQ ID NO: 1 and which provide reduced product inhibition. In other embodiments of the present invention, the cobalamin-dependent methionine synthases with reduced product inhibition in accordance with the present invention have at least one mutation in at least one of the following sequences:
wherein
X1=S, V, R; X2=S, E, R; X3=E, A, Q; X4=F, L, V; X5=L, R, S; X6=D, E, A, K; X7=A, Q, L; X8=A, N, S; X9=N, T, E; X10=H, R
wherein
X1=V, I; X2=V, L; X3=I, V, L; X4=g, A, L; X5=A, G; X6=M, Y; X7=Q, M, E; X8=L, I, F; X9=Q, M; X10=G, A, S, K; X11=F, Q, Y
wherein
X1=L, P, Y; X2=D, T, N; X3=V, L, D, E; X4=E, D, A, L; X5=K, D, P; X6=F except in T. marina, C. glutamicum, S. coel.; X7=R except in T. marina, C. glutamicum, S. coel.; X8=G except in T. marina, C. glutamicum, S. coel.; X9=E except in T. marina, C. glutamicum, S. coel.; X10=R except in T. marina, C. glutamicum, S. coel.; X11=F except in T. marina, C. glutamicum, S. coel.; X12=A except in T. marina, C. glutamicum, S. coel.; X13=D except in T. marina, C. glutamicum, S. coel.; X14=D, W except in T. marina; X15=F, P except in T. marina
wherein
X1=N, V; X2=D, L, I; X3=T, S, K; X4=R, K, A; X5=D, E; X6=V, I; X7=L, V, I; X8=R, A, L; X9=Q, S, A, K; X10=I, V; X11=R, E, N; X12=A, E, S; X13=F, I;
wherein
X1=E, A; X2=A, S; X3=A, V, S; X4=L, C, I, V; X5=V, I; X6=E, L; X7=G, N; X8=C, A, S; X9=N, T;
wherein
X1=L, H, T, R; X2=P, S, I, M; X3=N, A, K; X4=L. M; X5=A, G, R; X6=D, E, K; X7=Y, H; X8=D, Q, G; X9=I, M, L;
wherein
X1=A, P, E; X2=D, E, S; X3=R, L, K; X4=C, V, S, L; X5=R, H, A, D; X6=E, P; X7=L, I; X8=A, S, N, V; X9=Y, E, f, R; X10=K, A, N; X11=G, A; X12=T, A, V; X13=A, R, K; X14=V, L, I; X15=E, A, R; X16=V, C, A; X17=D, E; X18=F, M, W, K
wherein
X1=G except in T. marina, A, R except in T. marina; X2=R, T except in T. marina; X3=N, D, P except in T. marina; X4=G, G, E except in T. marina; X5=M, R, K except in T. marina; X6=R, Q, P except in T. marina; X7=F, W, Y except in T. marina;
wherein
X1=V, L, A, F; X2=S, V, D; X3=L, M, I; X4=G, T; X5=T, N, G; X6=K, R, E; X7=L, T; X8=P, A; X9=S, T, Y;
wherein
X1=F, Y; X2=>, T, D, E; X3=D, V, G, E; X4=L, F; X5=R, V, Y; X6=G, D, A, E; X7=H, A, N; X8=Y, E; X9=K, Q, R; X10=E, R; X11=A, N, S, T; X12=A, T, V; X13=L, E, K; X14=G, A, I; X15=I, L, M; X16=I, V; X17=E, A, E; X18=G, E; X19=G, A, V
wherein
X1=A, L, G; X2=F, L, I; X3=L, I; X4=I, V, F
wherein
X1=Q, F, S; X2=L, T, I; X3=Q, N, E; X4=V, T, A, L; X5=A, S; X6=H, L, F; X7=G, A; X8=V, A; X9=Q, R, K
wherein
X1=L, V; X2=D, G, S; X3=T, L, V, R; X4=F, D, E; X5=L, V; X6=P, F; X7=I, L; X8=I, M; X9=C, V, I, A; X10=H, S; X11=V, G, M
wherein
X1=V, I, F; X2=E, T, D; X3=T, D, E; X4=T, A, K; X5=G, S; X6=G except in T. marina, C. glutamicum, S. coel.; X7=T, R; X8=M, T, S; X9=M, L, S, T; X10=S, Q, T; X11=E, T, D
wherein
X1=G, E, A; X2=A, N; X3=A, F; X4=L, Y, A; X5=T, N, I; X6=A, S, T; X7=L, F
wherein
X1=P, H, E; X2=L, A; X3=G, E, D; X4=I, A; X5=D, L; X6=M, T, A; X7=I, F, L; X8=L, I; X9=A, S; X10=T, L; X11=D, A, E
In the context of the present invention the term “mutation” as regards an amino acid sequence relates to an amino acid substitution, insertion or deletion in the wild type sequence of cobalamin-dependent methionine synthases or functional fragments thereof with the requirement that the mutation changes the enzymatic activity such that the resulting polypeptide is still capable of catalyzing transfer of a methyl group from methyltetrahydrofolate to homocysteine in a cobalamin-dependent manner, with the mutated enzyme or functional fragment thereof having reduced product inhibition by methionine as defined above.
The person skilled in the art will be able to introduce mutations e.g. in the aforementioned amino acid sequences SEQ ID NOs: 1 to 18 and, e.g. relying on the assay described above, will be able to determine whether the resulting polypeptides are enzymatically active and show reduced product inhibition in the presence by methionine.
For such mutations, the person skilled in the art will consider in particular non-conservative amino acid substitutions, meaning that the wild-type amino acid is replaced with an amino acid of different physical-chemical properties. For example, if the wild-type sequence comprises a charged amino acid such as aspartate, a non-conservative substitution will include a substitution of the aspartate for a positively charged amino acid such as lysine. Alternatively, a negatively charged amino acid such as aspartic acid or glutamic acid may be replaced by a neutral amino acid such as glutamine, arginine or methionine. The person skilled in the art will, of course, also consider conservative amino acid substitutions, i.e. replacement by amino acids with comparable physico-chemical properties. An example is a replacement of Valine by Leucine.
An enzymatically active cobalamin-dependent methionine synthase or functional fragment thereof which carries a mutation in comparison to the respective wild type sequences is not considered to be polypeptide in accordance with the invention if it does not show a reduced product inhibition by methionine as it can be determined by the above mentioned test. Mutated polypeptides are also not considered to form part of the invention if they are not enzymatically active. This also applies for the nucleotide sequence encoding such polypeptides.
The nomenclature used throughout this specification for amino acids is the common one letter code.
As regards SEQ ID NOs: 3 to 18, “mutation” in the case of amino acid substitution means that the amino acid of a specified position can be replaced with any amino acid which is not specified for this particular position. For example, for SEQ ID NO: 3, X1 is specified to be S, V or R. A mutation may therefore comprise any amino acid substitution which is not S, V, R. Similarly, residue X4 of SEQ ID NO: 4, which is G, A, L, may be replaced by any amino acid which is not G, A, L.
The person skilled in the art is well aware that for any type of amino acid substitution, deletion or insertion, it will be necessary to determine whether the resulting polypeptide is (i) enzymatically active and (ii) shows a reduced product inhibition of the enzymatic activity in the presence of methionine.
The above explanations of the term “mutation” as given for amino acid sequences correspondingly apply for nucleotide and preferably DNA sequences encoding such polypeptides.
Specific embodiments of the present invention in case of the cobalamin-dependent methionine synthase MetH of C. glutamicum include mutations in positions 33, 86 and 134, wherein the wild-type sequence residues are methionine, phenylanaline and serine, respectively.
In the case of position 33, the methionine may be changed to glycine or alanine. In case of the phenylalanine 86 position, phenylalanine may be changed into leucine. In case of the serine residue at position 134, the residue may be changed into asparagine. The corresponding amino acid sequences are depicted in SEQ ID NOs: 19 to 22 respectively, while the corresponding DNA sequences are depicted in SEQ ID NOs: 25 to 28.
The person skilled in the art will realise that corresponding mutations may be introduced in e.g. the methionine residue 34 of the S. coel enzyme, the methionine residue 22 of the E. coli enzyme and the tyrosine residue in position 22 of the Thermotoga enzyme. As regards the phenylalanine 86 position of the C. glutamicum enzyme, corresponding mutations in the E. coli system would be located at the phenyl alanine residue 91, in the S. coel enzyme at the phenylalanine residue 86 and in the Thermotoga maritimum enzyme at the phenylalanine residue 76.
As regards the serine 134 residue in the C. glutamicum enzyme, corresponding mutations in the S. coel enzyme would be located at serine 134, in the E. coli enzyme at the valine residue 131 and in the Thermotoga enzyme at the aspartate residue 124.
In these cases, the corresponding residues may be mutated into glycine and alanine for the methione/tyrosine residues, into leucine for the phenylalanine residue and into asparagine for the serine, valine or aspartate residue.
Correspondingly, instead of amino acid substitution, cobalamin-dependent methionine synthases with reduced product inhibition in accordance with the present invention may have deletions at the aforementioned positions, or additional insertions.
Other embodiments of the present invention are nucleotide sequences and particularly DNA sequences which encode the aforementioned polypeptides and proteins. Some embodiments of the present invention relate to such DNA sequences in an isolated form.
Other embodiments of the present invention relate to vectors which comprise in 5′-3′ direction:
According to the present invention, operative linkage of a promoter, a nucleotide sequence in accordance with the present invention and a termination sequence means that nucleotide sequences in accordance with the present invention can be expressed in a host cell such that the host cell expresses an enzymatically active cobalamin-dependent methionine synthase or a functional fragment thereof that shows the reduced product inhibition by methionine as defined above.
In a preferred embodiment, these vectors comprise certain promoters and optionally enhancer elements to allow for over-expression of e.g. DNA sequences encoding the aforementioned polypeptides and proteins. Specific embodiments for expression and over-expression of DNA sequences are explained below.
Accordingly, another embodiment of the present invention relates to host cells which comprise nucleotide and preferably DNA sequences or vectors, as have been described above.
By genetically amending organisms in accordance with the present invention, the efficiency and/or yield and/or amount of methionine synthesis may be increased such that these methionine-overproducing organisms are characterized in that methionine is produced with an increased efficiency and/or increased yield and/or increased amount of preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90% or at least 100% compared to an initial organism that does not express the nucleotide sequences in accordance with the present invention.
Compared to such an initial host organism, the efficiency and/or yield and/or amount of methionine production in the methionine-producing host organism according to the present invention can increased preferably by at least by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 70, 100, 200, 500 or at least by a factor of 1000.
The host organism according to the present invention may be selected from the group consisting of coryneform bacteria, mycobacteria, streptomycetes, Salmonella, Escherichia coli, Shigella, Bacillus, Serratia, Pseudomonas, S. coel or T. maritima.
The organisms of the present invention may preferably comprise a microorganism of the genus Corynebacterium, particularly Corynebacterium acetoacidophilum, C. acetoglutamicum, C. acetophilum, C. ammoniagenes, C. glutamicum, C. lilium, C. nitrilophilus or C. spec.
The organisms in accordance with the present invention also comprise members of the genus Brevibacterium, such as Brevibacterium harmoniagenes, Brevibacterium botanicum, B. divaraticum, B. flavam, B. healil, B. ketoglutamicum, B. ketosoreductum, B. lactofermentum, B. linens, B. paraphinolyticum and B. spec.
The organisms in accordance with the present invention also comprise S. coel.
The organisms in accordance with the present invention also comprise members of the genus Thermotoga, such as T. maritime.
In particular, Corynebacterium microorganisms may be selected from the group consisting of Corynebacterium glutamicum (ATCC 13032), Corynebacterium acetoglutamicum (ATCC 15806), Corynebacterium acetoacidophilum (ATCC 13870), Corynebacterium thermoaminogenes (FERM BP-1539), Corynebacterium melassecola (ATCC 17965), Corynebacterium glutamicum (KFCC 10065), Corynebacterium glutamicum (DSM 17322), Corynebacterium efficiens (YS 314) and Corynebacterium glutamicum (ATCC21608).
Particularly preferred is the strain Corynebacterium glutamicum ATCC13032 and all its derivatives. The strains ATCC 13286, ATCC 13287, ATCC 21086, ATCC 21127, ATCC 21128, ATCC 21129, ATCC 21253, ATCC 21299, ATCC 21300, ATCC 21474, ATCC 21475, ATCC 21488, ATCC 21492, ATCC 21513, ATCC 21514, ATCC 21515, ATCC 21516, ATCC 21517, ATCC 21518, ATCC 21528, ATCC 21543, ATCC 21544, ATCC 21649, ATCC 21650, ATCC 21792, ATCC 21793, ATCC 21798, ATCC 21799, ATCC 21800, ATCC 21801, ATCC 700239, ATCC 21529, ATCC 21527, ATCC 31269 and ATCC 21526 which are known to produce lysine can also preferably be used. The other aforementioned strains can also be used.
The abbreviation KFCC means Korean Federation of Culture Collection, while the abbreviation ATCC means the American Type Strain Culture Collection Collection. The abbreviation DSM means the German Resource Centre for Biological Material.
Microorganisms of the genus Escherichia may be selected from the group comprising Escherichia coli. Microorganisms of the genus Salmonella may be selected from the group comprising Salmonella typhimurium.
Such host organisms may be engineered by introducing exogenous nucleotide sequences in accordance with the present invention, e.g. in the form of vectors.
In addition, or alternatively, mutations such as those described above which effect a reduced product inhibition may be introduced into the endogenous coding sequences for cobalamin-dependent methionine synthases.
A further embodiment of the present invention relates to host cells in which nucleotide and preferably DNA sequences in accordance with the present invention which encode for enzymatically active cobalamin-dependent methionine synthases or functional fragments thereof having a reduced product inhibition in the presence of methionine are expressed and in which additionally the endogenous gene(s) for cobalamin-dependent methionine synthase(s) is/are deleted or functionally disrupted.
The term “deleted” or “functional disruption” are, for the purposes of the present invention, equivalent to the statement that the content and/or activity of the cobalamin-dependent methionine synthases as they are encoded by the endogenous genes of the host organism are reduced.
How a reduction of the content and/or the activity and correspondingly a deletion and/or functional disruption of these endogenous genes for cobalamin-dependent methionine synthases may be achieved is described below.
Other embodiments of the present invention relate to host cells in which DNA sequences in accordance with the present invention, i.e. encoding cobalamin-dependent methionine synthases or fragments thereof with a reduced product inhibition in the presence of methionine are expressed and in which the content and/or activity of at least one of the following nucleotide sequences of group I is increased in comparison to the respective initial organism:
Of course, such host organisms may show an increased content and/or activity of nucleotide sequences which show a significant homology as defined above for any of the aforementioned nucleotide sequences. Again, the term “significant sequence homology” requires that these nucleotide sequences encode polypeptides that have the respective enzymatic activity.
In other embodiments of the present invention, the host organism may comprise nucleotide and preferably DNA sequences in accordance with the invention which encode cobalamin-dependent methionine synthases or active fragments thereof with reduced product inhibition in the presence of methionine and additionally provides a reduced content and/or activity of at least one of the following nucleotide sequences of group II:
The increase and/or decrease in the content and/or activity of the aforementioned nucleotide sequences of group I and II in comparison to the respective wild-type or initial organism may amount to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90% or at least 100%. Compared to such initial host organisms, the increase and/or decrease in the content and/or activity of the aforementioned nucleotide sequences of group I and II according to the present invention can amount also preferably to at least at factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 70, 100, 200, 500 or at least a factor of 1000.
Thus, all host cells and organisms in accordance with the present invention are characterized in that they comprise and preferably over-express at least one nucleotide and preferably DNA sequence in accordance with the present invention which encodes a protein or polypeptide that is an enzymatically active cobalamin-dependent methionine synthase or functional fragment thereof showing a reduced product inhibition in the presence of methionine as defined above. These polypeptides or proteins will usually carry at least one mutation in their amino acid sequence in comparison with the respective wild-type amino acid sequence which is responsible for the reduced product inhibition by methionine. In addition, these host cells may show an increase and/or decrease in the content and/or activity of any of the nucleotide sequences as specified above for group I and group II.
Alternatively, or in addition, these host cells may show a deletion or functional disruption of the endogenous genes encoding for wild-type cobalamin-dependent methionine synthases. Such host cells may be selected from the organisms specified above and produced in accordance with the methods described below.
Other embodiments of the present invention relate to methods for producing methionine in a host cell, wherein at least one nucleotide and preferably DNA sequence in accordance with the present invention, i.e. a sequence that encodes a polypeptide or protein which is an enzymatically active cobalamin-dependent methionine synthase or functional fragment thereof with reduced product inhibition in the presence of methionine as a consequence of a mutation in the amino acid sequence, is expressed in a host organism. Such host cells may be cultivated under appropriate conditions and the produced methionine be recovered. Such methods may also provide an increased efficiency and/or yield of methionine in comparison to the respective starting organism.
Other embodiments of the present invention relate to the use of nucleotide and preferably DNA sequences in accordance with the present invention and host cells in accordance with the present invention for producing methionine and/or increasing the efficiency and/or yield of methionine production.
With respect to increasing or decreasing the amount and/or activity of nucleotide sequences and the polypeptides being encoded thereby, all methods that are known in the art for increasing or decreasing the amount and/or activity of nucleotide sequence and/or a polypeptide in a host such as the above-mentioned organisms may be used. These methods are described in further detail below. These methods may also be used to express a DNA sequence in accordance with the present invention, i.e. a DNA sequence encoding a cobalamin-dependent methionine synthase with reduced product inhibition in the presence of methionine as a consequence of a mutation in the amino acid sequence.
Increasing or Introducing the Amount and/or Activity of Nucleotide Sequences and/or Polypeptides in Accordance with the Invention and of Group I
With respect to increasing the amount, two basic scenarios can be differentiated. In the first scenario, the amount of the polypeptide is increased by expression of an exogenous version of the respective nucleotide sequence. In the other scenario, expression of the endogenous polypeptide is increased by influencing the activity of promoter and/or enhancer elements and/or other regulatory activities such as phosphorylation, sumoylation, ubiquitylation, etc. that regulate the activities of the respective polypeptides either on a transcriptional, translational or post-translational level.
Besides simply increasing the amount of e.g. nucleotide sequences mentioned above, the activity of the polypeptides of e.g. group I may be increased by using enzymes carrying specific mutations that allow for an increased activity of the enzyme. Such mutations may, e.g. inactivate the regions of an enzyme that are responsible for feedback inhibition. By mutating these by e.g. introducing non-conservative mutations, the enzyme would not provide for feedback regulation anymore and thus activity of the enzyme would not be down regulated if more product was produced. The mutations may be either introduced into the endogenous copy of the enzyme, or may be provided by over-expressing a corresponding mutant form of the exogenous enzyme. Such mutations may comprise point mutations, deletions or insertions. Point mutations may be conservative or non-conservative. Furthermore, deletions may comprise only two or three amino acids up to complete domains of the respective protein. Of course, polypeptides in accordance with the invention, i.e. cobalamin-dependent methionine synthases or functional fragments thereof with reduced product inhibition can be expressed by expression of corresponding exogenous nucleotide sequences or by introducing the mutation(s) that leads to the reduced product inhibition in the endogenous genes.
Thus, the increase of the activity and the amount of a polypeptide may be achieved via different routes, e.g. by switching off inhibitory regulatory mechanisms at the transcription, translation, and protein level or by increase of gene expression of a nucleic acid coding for these proteins in comparison with the starting organism, e.g. by manipulating the endogenous gene or by introducing nucleic acids coding for the polypeptide.
In one embodiment, the increase of the activity and amount of a polypeptide, respectively, in comparison with the initial organism is achieved by an increase in the expression of a nucleic acid encoding such polypeptides. Sequences may be obtained from the respective database, e.g. at NCBI (HyperTextTransferProtocol://WorldWideWeb.ncbi.nlm.nihDOTgov/), EMBL (HyperTextTransferProtocol://WorldWideWeb.emblDOTorg), or Expasy (HyperTextTransferProtocol://WorldWideWeb.expasyDOTorg/), wherein “HyperTextTransferProtocol”=“http”, “WorldWideWeb”=“www”, and “DOT”=“.”.
In a further embodiment, the increase of the amount and/or activity of the above mentioned polypeptides is achieved by introducing the corresponding nucleic acids into the organism, preferably C. glutamicum, E. coli, S. coel or T. maritima.
In principle, every protein of different organisms with an enzymatic activity of the polypeptides mentioned above can be used. With genomic nucleic acid sequences of such enzymes from eukaryotic sources containing introns, already processed nucleic acid sequences like the corresponding cDNAs are to be used in the case that the host organism is not capable or cannot be made capable of splicing the corresponding mRNAs. All nucleic acids mentioned in the description can be, e.g., an RNA, DNA or cDNA sequence.
In one method according to the present invention for producing methionine, a nucleic acid sequence coding for one of the above-defined cobalamin-dependent methionine synthases or functional fragments thereof with reduced product inhibition by methionine is transferred to a microorganism such as C. glutamicum, E. coli, S. coel or T. maritima, respectively. This transfer leads to an increase in the expression of the mutated enzyme, and correspondingly to increased methionine synthesis.
According to the present invention, increasing and/or introducing the amount and/or the activity of a polypeptide typically comprises the following steps:
a) production of a vector comprising the following nucleic acid sequences, preferably DNA sequences, in 5′-3′-orientation:
b) transfer of the vector from step a) to the organisms of the invention such as C. glutamicum, E. coli, S. coel or T. maritima and, optionally, integration into the respective genomes.
The use of such vectors comprising regulatory sequences, like promoter and termination sequences are, is known to the person skilled in the art. Furthermore, the person skilled in the art knows how a vector from step a) can be transferred to organisms such as C. glutamicum, E. coli, S. coel or T. maritima and which properties a vector must have to be able to be integrated into their genomes.
If the enzyme content in an organism such as C. glutamicum is increased by transferring a nucleic acid coding for a polypeptide from another organism, like e.g. E. coli, it is advisable to transfer the amino acid sequence encoded by the nucleic acid sequence e.g. from E. coli by back-translation of the polypeptide sequence according to the genetic code into a nucleic acid sequence comprising mainly those codons, which are used more often due to the organism-specific codon usage. The codon usage can be determined by means of computer evaluations of other known genes of the relevant organisms.
According to the present invention, an increase of the gene expression and of the activity, respectively, of a nucleotide sequence in accordance with the present invention is also understood to be the manipulation of the expression of the endogenous respective endogenous enzymes of an organism, in particular of C. glutamicum, E. coli, S. coel or T. martima. This can be achieved, e.g., by altering the promoter DNA sequence for genes encoding, e.g. cobalamin-dependent methionine synthases with reduced product inhibition. Such an alteration, which causes an altered, preferably increased, expression rate of these mutated enzymes can be achieved by deletion or insertion of DNA sequences. Of course, this requires that mutations which are responsible for the reduced product inhibition have been introduced into the endogenous genes.
An alteration of the promoter sequence of such mutated endogenous genes usually causes an alteration of the expressed amount of the gene and therefore also an alteration of the activity detectable in the cell or in the organism.
Furthermore, an altered and increased expression, respectively, of an endogenous gene can be achieved by a regulatory protein, which does not occur in the transformed organism, and which interacts with the promoter of these genes. Such a regulator can be a chimeric protein consisting of a DNA binding domain and a transcription activator domain, as e.g. described in WO 96/06166.
A further possibility for increasing the activity and the content of endogenous genes is to up-regulate transcription factors involved in the transcription of the endogenous genes, e.g. by means of overexpression. The measures for overexpression of transcription factors are known to the person skilled in the art.
Furthermore, an alteration of the activity of endogenous genes can be achieved by targeted mutagenesis of the endogenous gene copies.
An alteration of endogenous genes coding for the enzymes of e.g. group I can also be achieved by influencing the post-translational modifications of the enzymes. This can happen e.g. by regulating the activity of enzymes like kinases or phosphatases involved in the post-translational modification of the enzymes by means of corresponding measures like overexpression or gene silencing.
In another embodiment, polypeptides of e.g. group I may be improved in efficiency, or its allosteric control region destroyed such that feedback inhibition of production of the compound is prevented. Similarly, a degradative enzyme may be deleted or modified by substitution, deletion, or addition such that its degradative activity is lessened for the desired polypeptides or the polypeptides being encoded by the nucleotide sequences of the present invention without impairing the viability of the cell. In each case, the overall yield or rate of production of methionine may be increased.
It is also possible that such alterations in the polypeptides and nucleotide molecules may improve the production of other fine chemicals such as other sulfur containing compounds like cysteine or glutathione, other amino acids, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose. Metabolism of any one compound can be intertwined with other biosynthetic and degradative pathways within the cell, and necessary cofactors, intermediates, or substrates in one pathway are likely supplied or limited by another such pathway. Therefore, by modulating the activity of polypeptides of the present invention and/or those of e.g. group I, the production and/or efficiency of another fine chemical biosynthetic or degradative pathway besides those leading to methionine may be impacted.
Enzyme expression and function may also be regulated based on the cellular levels of a compound from a different metabolic process, and the cellular levels of molecules necessary for basic growth, such as amino acids and nucleotides, may critically affect the viability of the microorganism in large-scale culture. Thus, modulation of an amino acid biosynthesis enzyme of e.g. the lysine biosynthetic pathways such that they are no longer responsive to feedback inhibition or such that they are improved in efficiency or turnover may result in better methionine production. The aforementioned strategies for increasing or introducing the amount and/or activity of the polypeptide and nucleotide sequences are not meant to be limiting; variations on these strategies will be readily apparent to one of ordinary skill in the art.
Reducing the Amount and/or Activity of Nucleotides Sequences and/or Polypeptides Encoding Endogenous Cobalamin Dependent Methionine Synthases and of Group II
For reducing the amount and/or activity of nucleotide sequence and polypeptides being encoded thereby, various strategies are also available.
The expression of the endogenous enzymes of e.g. group II can e.g. be regulated via the expression of aptamers specifically binding to the promoter sequences of the genes. Depending on the aptamers binding to stimulating or repressing promoter regions, the amount and thus, in this case, the activity of such enzymes is increased or reduced.
Aptamers can also be designed in a way as to specifically bind to the enzymes themselves and to reduce the activity of the enzymes by e.g. binding to the catalytic center of the respective enzymes. The expression of aptamers is usually achieved by vector-based overexpression (see above) and is, as well as the design and the selection of aptamers, well known to the person skilled in the art (Famulok et al., (1999) Curr Top Microbiol Immunol., 243, 123-36).
Furthermore, a decrease of the amount and the activity of e.g. the endogenous genes of MetH or of the endogenous enzymes of Group II can be achieved by means of various experimental measures, which are well known to the person skilled in the art. These measures are usually summarized under the term “gene silencing”. For example, the expression of an endogenous gene can be silenced by transferring an above-mentioned vector, which has a DNA sequence coding for the enzyme or parts thereof in antisense order, to the organisms such as C. glutamicum and E. coli. This is based on the fact that the transcription of such a vector in the cell leads to an RNA, which can hybridize with the mRNA transcribed by the endogenous gene and therefore prevents its translation.
Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Trends in Genetics, Vol. 1 (1) 1986.
In principle, the antisense strategy can be coupled with a ribozyme method. Ribozymes are catalytically active RNA sequences, which, if coupled to the antisense sequences, cleave the target sequences catalytically (Tanner et al., (1999) FEMS Microbiol Rev. 23 (3), 257-75). This can enhance the efficiency of an antisense strategy.
Further methods are the introduction of nonsense mutations into the endogenous gene by means of introducing RNA/DNA oligonucleotides into the organism (Zhu et al., (2000) Nat. Biotechnol. 18 (5), 555-558) or generating knockout mutants with the aid of homologous recombination (Hohn et al., (1999) Proc. Natl. Acad. Sci. USA. 96, 8321-8323.).
To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of gene coding for e.g. an enzyme of group II or the endogenous MetH gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the endogenous gene.
Preferably, this endogenous gene is a C. glutamicum or E. coli gene, but it can be a homologue from a related bacterium or even from a yeast or plant source. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i. e. no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous enzyme of e.g. group 2). In the homologous recombination vector, the altered portion of the endogenous gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the endogenous gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in the (micro)organism. The additional flanking endogenous nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas, K. R., and Capecchi, M. R. (1987) Cell 51: 503 for a description of homologous recombination vectors).
The vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced endogenous gene has homologously recombined with the endogenous enzymes are selected, using art-known techniques.
In another embodiment, an endogenous gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional enzyme. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an endogenous gene in a (micro)organism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the endogenous gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the genes coding e.g. for the enzymes of group II and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.
Furthermore, a gene repression (but also gene overexpression) is also possible by means of specific DNA-binding factors, e.g. factors of the zinc finger transcription factor type. Furthermore, factors inhibiting the target protein itself can be introduced into a cell. The protein-binding factors may e.g. be the above-mentioned aptamers (Famulok et al., (1999) Curr Top Microbiol Immunol. 243, 123-36).
As further protein-binding factors, whose expression in organisms cause a reduction of the amount and/or the activity of the enzymes of e.g. group II, enzyme-specific antibodies may be considered. The production of monoclonal, polyclonal, or recombinant enzyme-specific antibodies follows standard protocols (Guide to Protein Purification, Meth. Enzymol. 182, pp. 663-679 (1990), M. P. Deutscher, ed.). The expression of antibodies is also known from the literature (Fiedler et al., (1997) Immunotechnology 3, 205-216; Maynard and Georgiou (2000) Annu. Rev. Biomed. Eng. 2, 339-76).
The mentioned techniques are well known to the person skilled in the art. Therefore, he also knows which sizes the nucleic acid constructs used for e.g. antisense methods must have and which complementarity, homology or identity, the respective nucleic acid sequences must have. The terms complementarity, homology, and identity are known to the person skilled in the art.
The term complementarity describes the capability of a nucleic acid molecule of hybridizing with another nucleic acid molecule due to hydrogen bonds between two complementary bases. The person skilled in the art knows that two nucleic acid molecules do not have to have a complementarity of 100% in order to be able to hybridize with each other. A nucleic acid sequence, which is to hybridize with another nucleic acid sequence, is preferred being at least 30%, at least 40%, at least 50%, at least 60%, preferably at least 70%, particularly preferred at least 80%, also particularly preferred at least 90%, in particular preferred at least 95% and most preferably at least 98 or 100%, respectively, complementary with said other nucleic acid sequence.
Nucleic acid molecules are identical, if they have identical nucleotides in identical 5′-3′-order.
The hybridization of an antisense sequence with an endogenous mRNA sequence typically occurs in vivo under cellular conditions or in vitro. According to the present invention, hybridization is carried out in vivo or in vitro under conditions that are stringent enough to ensure a specific hybridization.
Stringent in vitro hybridization conditions are known to the person skilled in the art and can be taken from the literature (see e.g. Sambrook et al., Molecular Cloning, Cold Spring Harbor Press). The term “specific hybridization” refers to the case wherein a molecule preferentially binds to a certain nucleic acid sequence under stringent conditions, if this nucleic acid sequence is part of a complex mixture of e.g. DNA or RNA molecules.
The term “stringent conditions” therefore refers to conditions, under which a nucleic acid sequence preferentially binds to a target sequence, but not, or at least to a significantly reduced extent, to other sequences.
Stringent conditions are dependent on the circumstances. Longer sequences specifically hybridize at higher temperatures. In general, stringent conditions are chosen in such a way that the hybridization temperature lies about 5° C. below the melting point (Tm) of the specific sequence with a defined ionic strength and a defined pH value. Tm is the temperature (with a defined pH value, a defined ionic strength and a defined nucleic acid concentration), at which 50% of the molecules, which are complementary to a target sequence, hybridize with said target sequence. Typically, stringent conditions comprise salt concentrations between 0.01 and 1.0 M sodium ions (or ions of another salt) and a pH value between 7.0 and 8.3. The temperature is at least 30° C. for short molecules (e.g. for such molecules comprising between 10 and 50 nucleotides). In addition, stringent conditions can comprise the addition of destabilizing agents like e.g. form amide. Typical hybridization and washing buffers are of the following composition.
A typical procedure for the hybridization is as follows:
These stringent conditions also apply as far as the claims relate to DNA sequences that hybridise under stringent conditions.
The terms “sense” and “antisense” as well as “antisense orientation” are known to the person skilled in the art. Furthermore, the person skilled in the art knows, how long nucleic acid molecules, which are to be used for antisense methods, must be and which homology or complementarity they must have concerning their target sequences.
Accordingly, the person skilled in the art also knows, how long nucleic acid molecules, which are used for gene silencing methods, must be. For antisense purposes complementarity over sequence lengths of 100 nucleotides, 80 nucleotides, 60 nucleotides, 40 nucleotides and 20 nucleotides may suffice. Longer nucleotide lengths will certainly also suffice. A combined application of the above-mentioned methods is also conceivable.
If, according to the present invention, DNA sequences are used, which are operatively linked in 5′-3′-orientation to a promoter active in the organism, vectors can, in general, be constructed, which, after the transfer to the organism's cells, allow the overexpression of the coding sequence or cause the suppression or competition and blockage of endogenous nucleic acid sequences and the proteins expressed there from, respectively.
The activity of a particular enzyme may also be reduced by over-expressing a non-functional mutant thereof in the organism. Thus, a non-functional mutant which is not able to catalyze the reaction in question, but that is able to bind e.g. the substrate or co-factor, can, by way of over-expression out-compete the endogenous enzyme and therefore inhibit the reaction. Further methods in order to reduce the amount and/or activity of an enzyme in a host cell are well known to the person skilled in the art.
Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleotide sequence in accordance with the invention (or portions thereof) or combinations thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.
Such vectors are referred to herein as “expression vectors”.
In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
The recombinant expression vectors of the invention may comprise a nucleic acid in accordance with the present invention and/or coding for the enzymes of group I in a form suitable for expression of the respective nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, repressor binding sites, activator binding sites, enhancers and other expression control elements (e.g., terminators, polyadenylation signals, or other elements of mRNA secondary structure). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-, tet-, lpp-, lac-, lpp lac-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SP02, e-Pp-ore PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such asCaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids in accordance with the invention.
The recombinant expression vectors of the invention can be designed for expression of the polypeptides in accordance with the invention in prokaryotic or eukaryotic cells. For example, the genes for the enzymes of Group I can be expressed in bacterial cells such as C. glutamicum and E. coli, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M. A. et al. (1992), Yeast 8: 423-488; van den Hondel, C. A. M. J. J. et al. (1991) in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) Plant Cell Rep.: 583-586). Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315), pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-Bl, egtll, pBdC1, and pET lld (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89; and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: N.Y. IBSN 0 444 904018). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET lld vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7gnl). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) from a resident X prophage harboring a T7gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmidspIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB110, pC194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: N.Y. IBSN 0 444 904018).
One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
Examples of suitable C. glutamicum and E coli shuttle vectors can be found in Eikmanns et al (Gene. (1991) 102, 93-8).
In another embodiment, the protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J. 6: 229-234), 2i, pAG-1, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: N.Y. (IBSN 0 444 904018).
For the purposes of the present invention, an operative link is understood to be the sequential arrangement of promoter, coding sequence, terminator and, optionally, further regulatory elements in such a way that each of the regulatory elements can fulfill its function, according to its determination, when expressing the coding sequence.
For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003.
Another aspect of the invention pertains to organisms or host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection”, “conjugation” and “transduction” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e. g., linear DNA or RNA (e. g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003), and other laboratory manuals.
In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding polypeptides of the present invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e. g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of a nucleotide sequence of the present invention on a vector placing it under control of the lac operon permits expression of the gene only in the presence of IPTG. Such regulatory systems are well known in the art.
In one embodiment, the method comprises culturing the organisms of invention (into which a recombinant expression vector encoding e.g. a polypeptide of the present invention has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered enzyme) in a suitable medium for methionine production. In another embodiment, the method further comprises isolating methionine from the medium or the host cell.
Growth of Escherichia Coli and Corynebacterium Glutamicum-Media and Culture Conditions
The person skilled in the art is familiar with the cultivation of common microorganisms such as C. glutamicum and E. coli. Thus, a general teaching will be given below as to the cultivation of C. glutamicum. Corresponding information may be retrieved from standard textbooks for cultivation of E. coli.
E. coli strains are routinely grown in MB and LB broth, respectively (Follettie, M. T., Peoples, 0., Agoropoulou, C., and Sinskey, A J. (1993) J. Bacteriol. 175, 4096-4103). Minimal media for E. coli is M9 and modified MCGC (Yoshihama, M., Higashiro, K., Rao, E. A., Akedo, M., Shanabruch, W G., Follettie, M. T., Walker, G. C., and Sinskey, A. J. (1985) J. Bacteriol. 162, 591-507), respectively. Glucose may be added at a final concentration of 1%. Antibiotics may be added in the following amounts (micrograms per milliliter): ampicillin, 50; kanamycin, 25; nalidixic acid, 25. Amino acids, vitamins, and other supplements may be added in the following amounts: methionine, 9.3 mM; arginine, 9.3 mM; histidine, 9.3 mM; thiamine, 0.05 mM. E. coli cells are routinely grown at37 C, respectively.
Genetically modified Corynebacteria are typically cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32: 205-210; von der Osten et al. (1998) Biotechnology Letters, 11: 11-16; Patent DE 4,120,867; Liebl (1992) “The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al., eds. Springer-Verlag).
These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources.
It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH4Cl or (NH4)2SO4, NH4OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
The overproduction of methionine is possible using different sulfur sources. Sulfates, thiosulfates, sulfites and also more reduced sulfur sources like H2S and sulfides and derivatives can be used. Also organic sulfur sources like methyl mercaptan, thioglycolates, thiocyanates, and thiourea, sulfur containing amino acids like cysteine and other sulfur containing compounds can be used to achieve efficient methionine production. Formate may also be possible as a supplement as are other C1 sources such as methanol or formaldehyde. Particularly suited are methanethiol and its dimer dimethyldisulfide.
Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate-salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook “Applied Microbiol. Physiology, A Practical Approach (Eds. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components should be sterilized, either by heat (20 minutes at 1.5 bar and 121 C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately.
All media components may be present at the beginning of growth, or they can optionally be added continuously or batch wise. Culture conditions are defined separately for each experiment.
The temperature should be in a range between 15° C. and 45° C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium may be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH4OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100-300′rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.
If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD600 of 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2.5 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 agar, pH 6.8 with 2M NaOH) that had been incubated at 30 C.
Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.
The above culture and media conditions may also be applied for other host organisms such as S. coel and T. maritima.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06127363 | Dec 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/064471 | 12/21/2007 | WO | 00 | 6/25/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/080900 | 7/10/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7884066 | Ting | Feb 2011 | B2 |
| 7884069 | Schaebitz et al. | Feb 2011 | B2 |
| 7884263 | Dewey et al. | Feb 2011 | B2 |
| Number | Date | Country |
|---|---|---|
| 0210209 | Feb 2002 | WO |
| 02051231 | Jul 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100035307 A1 | Feb 2010 | US |
