Patent Application
20170335294
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled, “KRI0001_401_PC_Sequence_Listing_20150504”, created May 4, 2015, which is 104,033 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
Nitrogen fixation.
Nitrogen fixation is one of the key processes required for life on Earth. Nitrogen is an essential building block for basic biological molecules, such as DNA and proteins. While 78% of the Earth's atmosphere is comprised of nitrogen gas (N2), most organisms, plants and animals included, are unable to directly utilize atmospheric nitrogen for metabolic purposes as it must first be converted into a water soluble compound. In the nitrogen fixation process, molecular nitrogen is reduced to water-soluble form (for ex. ammonia), and becomes available for use by living organisms (Rees et al, Philos Transact A Math Phys Eng Sci, 2005, 363: 971-984).
In nature, one of the key sources of bioavailable nitrogen are diazotrophs, microorganisms converting atmospheric nitrogen into soluble nitrogenous compounds through the nitrogen fixation process. To increase nitrogen bioavailability, some plants (e.g. legumes) are known for their ability to establish symbiosis with nitrogen fixing microorganisms (e.g. rhizobia). Other natural sources of bioavailable nitrogen are known, such as fixation by lightning or decomposition of living matter. In agriculture, nitrogen is often provided to crops in the form of fertilizer generated chemically under high temperature and pressure through the Haber-Bosch process. Approximately 108 tons of nitrogen are fixed on an annual basis by the chemical industry to maintain appropriate levels of agricultural production to feed the growing world's population (Rees et al, Philos Transact A Math Phys Eng Sci, 2005, 363:971-984).
Plants are typically not capable of fixing nitrogen on their own and must rely on the aforementioned external supply sources of bioavailable nitrogen. In nature, nitrogenase is the enzyme responsible for biological fixation of nitrogen (Rees et al, Philos Transact A Math Phys Eng Sci, 2005, 363: 971-984; Cheng Q, J Int Plant Biol, 2008, 50(7):784-96). A variety of nitrogenases from different organisms are known in the art. Transgenic plants comprising a nitrogenase, and thus capable of fixing nitrogen on their own, would greatly enhance agricultural productivity and reduce costs due to decrease or elimination of nitrogen fertilizer use. The reduction of nitrogen fertilizer use will also decrease the harsh impacts of fertilizer run-offs on the environment and human health. Thus it would be of great economic and social benefit to generate plants capable of fixing nitrogen on their own.
For over a century, since the discovery of nitrogen fixation, myriads of scientists and laymen alike contemplated and prophesized about creating plants capable of nitrogen fixation using essentially any known biological mechanism and a nitrogenase system. However, as of today, no one has ever been able to create such plants.
In the mid 1990's, Meyer's group published a number of articles with an initial description of Streptomyces thermoautotrophicus nitrogenase system: (i) Gadkari et al, Appl Environ Microbiol, 1990, 56(12):3727-34; (ii) Gadkari et al, J Bacteriol, 1992, 174(21):6840-3; and (iii) Ribbe et al, J Biol Chem, 1997, 272(42):26627-33. However, these publications neither contemplated, demonstrated nor enabled the use of Streptomyces thermoautotrophicus nitrogenase in plants or plant cells. This work provided only initial and very limited information in regard to the biochemistry, biology, genetics or functionality of Streptomyces thermoautotrophicus nitrogenase, and shed no light on its compatibility to other biological systems.
Patent application US 2014/0011261, by Wang et al., prophetically contemplates the use of prokaryotic nif encoded nitrogenases, specifically driven by T7 promoter(s), in eukaryotic cells to hypothetically enable nitrogen fixation. Yet another application US 2014/0196178, by Zaltsman, also prophetically proposes the use of nif genes to generate plants capable of nitrogen fixation. These publications have no bearing or relation to the present invention as nif nitrogenase system and genes have no biochemical, mechanistic, genetic, evolutionary, or any other relation to Streptomyces thermoautotrophicus nitrogenase, which is well known in the art to be an exceptional and unusual mechanism for nitrogen fixation (for ex. see Giller and Mapfumo, Encyclopedia of Soil Science, 2006 by Taylor & Francis). Moreover, Streptomyces thermoautotrophicus and its nitrogenase system are not disclosed or contemplated in these prophetic applications.
Nitrogen fixation is a very broad field of science with a large amount of data collected. Hence there certainly are additional patents, applications and publications in this field, such as for example the work of Cocking (U.S. Pat. No. 7,470,427 and related art), contemplating the use of bacteria living intracellularly within plant cells to enable plants to fix nitrogen. However, these works, similarly to the other publications mentioned herein, have no relation or bearing on the present invention besides all being affiliated with the broad field of nitrogen fixation.
Importantly, within the past few years, Streptomyces thermoautotrophicus nitrogenase system came under strong skepticism in the art. For example, presenters at the 18th International Congress on Nitrogen Fixation in Miyazaki, Japan, suggested that heterologous expression of Streptomyces thermoautotrophicus nitrogenase only yields hydrogen production and not necessarily fixes nitrogen. This and other occurrences have led to a prevailing belief in the field—fueled by failures of others, which in the scientific community typically not published but rather communicated directly between scientists in meetings and conferences—that Streptomyces thermoautotrophicus nitrogenase is not fit for nitrogen fixation. Additional examples of such communications are well known to those skilled in the art, for instance, a presentation by a large group of scientists at the 11th European Nitrogen Fixation Conference entitled “The genome of Streptomyces thermoautotrophicus does not contain sequences of classical or non-classical nitrogenases and three independent isolates do not fix nitrogen” by Drew MacKellar and Pamela Silver of Harvard Medical School, Boston, Mass., USA; Tony Bolger, Bjorn Usadel and Jurgen Prell of RWTH Aachen University, Aaachen, Germany; Cory Tobin of California Institute of Technology, Calif., USA; James Murray and Bill Rutherford of Imperial College, London, UK; Lucas Lieber of Universidad Nacional de Rosario, Argentina; Jeffery Norman and Maren Friesen of Michigan State University, Mich., USA.
For many decades there was a long-felt and persistent need to create plants capable of nitrogen fixation. This concept has even been referred by those skilled in the art as the “holy grail” of agricultural biotechnology. Yet, despite multiple attempts well known to those skilled in the art, until the present invention, no one was able to create plants capable of nitrogen fixation.
The instant invention daringly defies currently accepted perception and state of the art in the field, showing that, unexpectedly and conversely to what is presently known to those skilled in the art, Streptomyces thermoautotrophicus nitrogenase can be used to fix nitrogen, and further can be used to generate plants capable of fixing nitrogen.
Nitrogenases are present in certain organisms in the nature, but not in plants. These enzymes are typically found in specialized organisms and tissues which evolved the capacity to carry out nitrogen fixation (Rees et al, Philos Transact A Math Phys Eng Sci, 2005, 363:971-984). No example, particularly of higher plants capable of nitrogen fixation on their own (e.g. without symbionts), is known in nature today.
For over a century many have dreamed, prophesized and speculated in regards to prospects and benefits of generating plants capable of nitrogen fixation on their own. Essentially all and any known nitrogen fixation system has been a subject to these desires and dreams. However, despite multiple efforts and constant trial, as of today no one has yet been able to create plants capable of fixing nitrogen on their own and bring this concept to reality.
The present invention has been uncovered as a surprising and unexpected result of an attempt to study and further characterize certain parts (St1 and St2, but not St3) of Streptomyces thermoautotrophicus nitrogenase system. Very limited data is available in regards to Streptomyces thermoautotrophicus nitrogenase as of today and additional information is needed to better understand its functionality. As described herein, chloroplast expression, amongst other expression systems considered, was preferred due to its capacity to produce large amounts of recombinant proteins at relatively low cost. This system can be used to express and purify Streptomyces thermoautotrophicus polypeptides of St1 and St2 complexes to study their properties, including purification and detection with anti-nitrogenase (S. thermoautotrophicus) antibodies, enzyme kinetics, functionality at different temperatures, prospective additional molecular partners and cofactors, crystallography studies, and many other aspects. It was not expected that St1 and St2 expressing plants would be able to fix nitrogen directly due to well-known skepticism in the art (see section “Related Art”) as well as further reasons detailed below.
In one embodiment, the instant invention encompasses plants and plant cells comprising Streptomyces thermoautotrophicus nitrogenase, and plants or plant cells capable of nitrogen fixation. In another embodiment, the present invention includes heterologous cells or organisms (e.g. other than Streptomyces thermoautotrophicus) comprising nitrogenase from Streptomyces thermoautotrophicus, and wherein said organisms can also become capable of nitrogen fixation. In yet another embodiment, the present invention includes nitrogenases from other species, particularly those carrying homologs of Streptomyces thermoautotrophicus nitrogenase, i.e. enzymes with homology to carbon monoxide dehydrogenases and having the capacity to fix nitrogen, as well as nitrogenases modified, improved or enhanced via mutagenesis, directed evolution, codon optimization or other methods known in the art. The current invention encompasses any plant, plant cell, heterologous cell or organism comprising Streptomyces thermoautotrophicus nitrogenase, wherein said nitrogenase bestows the trait of nitrogen fixation to said organism, cell or plant.
In one embodiment, the present invention demonstrates, and for the first time enables, a novel and highly advantageous method where a plant becomes capable of fixing nitrogen on its own through expression of nitrogenase components directly within a plant cell. In another embodiment, the instant invention contemplates nitrogenase expression in heterologous unicellular or multicellular organisms, other than plants, which are unable to fix nitrogen on their own, thus resulting in an unusual and novel trait of nitrogen fixation in said heterologous organisms. Also, nitrogenase can be expressed in such heterologous organisms for other reasons, such as study of nitrogenase and its functions in a new cellular environment or production of nitrogenase proteins for research and educational purposes. Non-limiting examples of said heterologous organisms are prokaryotes and eukaryotes including, but not limited to bacteria, cyanobacteria, archea, fungi, protists, algae or animals, which are naturally unable of fixing nitrogen.
In one aspect, the invention relates to a plant or a heterologous cell containing an expressible heterologous nucleotide sequence comprising a nitrogenase gene or genes, wherein the heterologous nucleotide sequence is expressed to render the plant or a heterologous cell capable of fixing nitrogen. In another aspect, the present invention relates to a method of producing a plant, heterologous cell, or organism capable of fixing nitrogen on its own. This method includes transfecting a plant cell or a heterologous cell with a vector comprising an expressible heterologous nucleotide sequence of a nitrogenase gene or genes.
Streptomyces thermoautotrophicus nitrogenase system can be employed for expression in a large number of biological systems, for either generating novel cells and organisms capable of nitrogen fixation, or for protein expression purposes to further research into its functionality, or for other studies. Non-limiting examples of such organisms include mycorrhizal fungi, which can be further used as biofertilizers, or bacterial cells such as E. coli which can be used as model organisms in research or protein expression and purification, or algal cells that can be used for biofuel production.
The present invention is instrumental for producing plants, including agriculturally important crops such as corn and cotton, with reduced or abolished requirements for nitrogen fertilizer, leading to reduced costs of agricultural production. In addition, this novel technology can produce multiple environmental benefits. Reduction in nitrogen fertilizer use will decrease incidence of fertilizer run-offs, helping to reduce negative impact on water supplies, wildlife and human health. Moreover, reduced nitrogen requirements may provide an important advantage to row crops over weeds and lead to reduced use of herbicides in agriculture, further decreasing impact on the environment and human health.
Amongst its many embodiments, the present disclosure encompasses:
The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
Those of ordinary skill in the art will recognize that any and all features, combinations of features, or permutations of features discussed or possible herein, including those in the description, figures, sequence listings, examples and claims, is (are) linked and are clearly and unambiguously intended to be included within the scope of the present disclosure and claims, provided that the features included in any such combination or permutation are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Thus, additional advantages and aspects of the present invention beyond those specifically described herein will be readily apparent and enabled to those of ordinary skill in the art from the entirety of the present disclosure and claims. The contents of all publications, patent applications, patents and other references mentioned herein are incorporated by reference herein in their entirety. In case of conflict, the present specification, including explanations of terms, will control.
The term “consisting” as used herein in a claim means that the invention necessarily includes the listed ingredients, but is opened to unlisted ingredients that do not materially affect the properties of the invention. The term “comprising” in a claim herein is open-ended and means that the claim must have all the features specifically recited herein, but there is no bar on additional features that are not recited, thus leaving the claim open for the inclusion of other unspecified features. The term “consisting essentially of” in a claim is an intermediate term between claims that are written in a “consisting” format and those drafted in an open “comprising” format. All these terms can be used interchangeably herein. The use of the term “including”, as well as other related forms such as “includes” and “included”, is not limiting.
“Control” or “control level” means the level of an enzymatic or biological activity normally or typically found in nature. A control level is also referred to as a wild type or base-line level. A control plant, i.e. a plant that does not contain the recombinant DNA that confers a particular trait (ex. nitrogen fixation capacity), is used as a baseline for comparison to identify or characterize said particular trait. A suitable control plant may be a non-transgenic wild-type plant, or it may also be a transgenic plant line that comprises an empty vector, a selection marker or a marker gene, but does not contain the recombinant DNA that encodes said particular trait (ex. nitrogen fixation capacity).
The term “about” as used herein is a word with a flexible meaning akin to “nearly”. The term “about” indicates that exactitude is not claimed, but rather a contemplated variation. Thus, as used herein, the term “about” means within one or two standard deviations from the recited value, or +/− a range of up to 50%, up to 25%, up to 10%, up to 5%, or up to 4%, 3%, 2% or 1% as compared to the recited value.
As used herein, the term “Streptomyces thermoautotrophicus nitrogenase” refers to the nitrogenase of the UBT1 strain of Streptomyces thermoautotrophicus described by Ribbe et al, J Biol Chem, 1997, 272(42):26627-33 (DSMZ 41605; ATCC 49746), as well as to the nitrogenases from subspecies, varieties, strains, accessions or other close taxonomic relatives of UBT1 having the same or similar (i.e., within the range of from about 50% to about 200%, or more) of the UBT1 nitrogenase ability to fix atmospheric N2. Similar subspecies, varieties, strains, accessions or other close relatives of UBT1 possessing such nitrogenases may include, for example, those disclosed in, but not limited to, Bergey's Manual of Systematic Bacteriology (Book 5), Springer-Verlag, second edition, 2012, pages 1554 and 1744; Kim et al, International Journal of Systematic Bacteriology, 1999, 49: 7-17; as well as accessions available from ATCC, DSM, DPDU, KCTC, NRRL, ISP, NCIM, CUB, IFO, IMSNN and other depositories or culture collections, as well as those exhibiting physical, biochemical, physiological and other characteristics consistent with those of strain UBT1 disclosed in Ribbe et al, J Biol Chem, 1997, 272(42):26627-33.
Plants and Plant Cells Comprising Streptomyces thermoautotrophicus Nitrogenase
This invention has been uncovered as a surprising and unexpected result during an attempt to study and further characterize Streptomyces thermoautotrophicus nitrogenase polypeptides. As of today, very limited data is available in regards to Streptomyces thermoautotrophicus nitrogenase system and additional information is needed to better understand its functionality. Nicotiana tabacum (tobacco) chloroplast expression system, known to produce large amounts of expressed transgenes at a very low cost, has been selected to express and purify Streptomyces thermoautotrophicus nitrogenase polypeptides (using, for example, monoclonal-antibody conjugated columns and other methods known in the art) and to study their properties, including but not limited to enzyme kinetics, stability, crystallography and other aspects. For brevity, here we demonstrate expression of Streptomyces thermoautotrophicus polypeptides in chloroplasts, which is given by way of illustration only and is not limitative of the presently disclosed embodiments. Expression of Streptomyces thermoautotrophicus polypeptides can also be achieved from nuclear or mitochondrial genomes, or episomal units, and combinations of any of the foregoing, using methods and technologies well known in the art and is not presented here for brevity.
For a host of reasons, in addition to those detailed in section “Related Art” above, it was not expected that plants carrying Streptomyces thermoautotrophicus nitrogenase would be able to fix nitrogen directly from the air. To name a few examples, Streptomyces thermoautotrophicus is an extremophile, thriving in physically extreme conditions that are detrimental to most life on Earth, and the functional temperature for its nitrogenase is 65° C. Plants function at much lower temperatures (normally 18-26° C.), at which Streptomyces thermoautotrophicus nitrogenase is not expected to work, i.e. to be functionally active in fixing of atmospheric nitrogen. Furthermore, the reaction is coupled to oxidation of carbon monoxide, found in Streptomyces thermoautotrophicus' natural environment, but not in conventional plant environments. Moreover, Streptomyces thermoautotrophicus nitrogenase is functionally dependent on St3 CO dehydrogenase (see Ribbe et al, J Biol Chem, 1997, 272(42):26627-33), found in very specific aerobic and anaerobic microbes, but which is not typical in plants, for supply of superoxide anion radicals. St3 CO dehydrogenase Cox proteins, an integral part of Streptomyces thermoautotrophicus nitrogenase system, was not expressed in the experiments with the nitrogenase parts St1 and St2 described herein, and hence the expressed partial nitrogenase complex was not expected to be functional in the absence of St3 CO dehydrogenase. Thus, according to the present invention, St1 and St2 are sufficient to catalyze nitrogen fixation in transgenic organisms such as plants. In addition, plants are extremely different in their biochemistry, genetics and other biological aspects from extremophiles like Streptomyces thermoautotrophicus. These broad biological differences manifest in vast differences in protein expression and post-translational modifications, stability, generation of correct ratio of protein complex subunits, availability of correct amounts of co-factors (Mo, Mn, Fe, etc.), and multiple other factors, rendering the possibility of Streptomyces thermoautotrophicus nitrogenase functionality in plants highly unlikely and unexpected. What's more, functional nitrogenase of Streptomyces thermoautotrophicus produces ammonia, which can be toxic to hosts such as plant cells and plants. While Streptomyces thermoautotrophicus have had an evolutionary opportunity to develop biological mechanisms to reduce toxic effects of ammonia, heterologous expression of functional Streptomyces thermoautotrophicus nitrogenase in plants was likely to result in significant cellular damage and death due to the sudden and unexpected appearance of ammonia in the cells. Thus, it was highly unexpected that plant cells could survive and thrive with a functional Streptomyces thermoautotrophicus nitrogenase.
It was noticed in the experiments herein that neglected transgenic Nicotiana tabacum plants comprising Streptomyces thermoautotrophicus nitrogenase had an extent of a different appearance than neglected wild-type plants or other transgenics. The slender difference in appearance might have resulted from differences in nutrient metabolism, and it was decided to investigate further. A very simple experiment of planting Nicotiana tabacum comprising Streptomyces thermoautotrophicus nitrogenase side by side with wild type Nicotiana tabacum on nitrogen free MSO medium was performed. Astoundingly, plants comprising Streptomyces thermoautotrophicus nitrogenase showed diminished signs of nitrogen deficiency as compared to wild type control plants, which was further determined to be a result of Streptomyces thermoautotrophicus nitrogenase activity, a highly unexpected and surprising result.
In one aspect, the present invention encompasses plants and plant cells comprising Streptomyces thermoautotrophicus nitrogenase enzyme or enzyme complex, rendering the transgenic plants capable of fixing nitrogen. In one embodiment, the plant or plant cell comprises components St1 and St2 of Streptomyces thermoautotrophicus nitrogenase (Ribbe et al, J Biol Chem, 1997, 272 (42):26627-33). Optionally, St3 component may be expressed in the same cell to enhance or modify nitrogenase activity. The nitrogenase complex of Streptomyces thermoautotrophicus, a free-living nitrogen fixing bacterium, catalyzes the following reaction:
N2+4-12MgATP+8H++8e−=2NH3+H2+4-12MgADP+4-12Pi
Streptomyces thermoautotrophicus in is an extremophile which has been isolated from the covering soil of burning charcoal piles. In the reaction mediated by its nitrogenase, oxidation of carbon monoxide is coupled by a molybdenum-containing CO dehydrogenase (CODH), resulting in transfer of electrons derived from CO oxidation to oxygen and producing O2 superoxide anion radicals. Reoxidation of the O2 superoxide anion radicals to molecular oxygen by Mn-containing superoxide oxidoreductase is followed by transfer of the electrons by a MoFeS-dinitrogenase to N2 and culminates in the production of ammonium ions (and ammonia) (Ribbe et al, J Biol Chem, 1997, 272 (42): 26627-33).
The complete Streptomyces thermoautotrophicus nitrogenase complex is comprised of components designated as St1, St2 and St3 (
The genes coding for the nitrogenase polypeptide components (SEQ ID NOs: 5-8) can be isolated from the genome of Streptomyces thermoautotrophicus UBT1 (DSMZ 41605; ATCC 49746) (Ribbe et al, J Biol Chem, 1997, 272 (42):26627-33), or other species carrying similar nitrogenase systems. As noted above, other similar nitrogenase genes can also be isolated from subspecies, varieties, strains, accessions, or other close relatives of UBT1 having the same or similar (i.e., within the range of from about 50% to about 200%, or more) of the UBT1 nitrogenase ability to fix atmospheric N2. Exemplary partial amino acid sequences of St1 and St2 components from Streptomyces thermoautotrophicus are shown in SEQ ID NOs: 1-4 (per Ribbe et al, J Biol Chem, 1997, 272 (42):26627-33). Exemplary full length DNA sequences of St1 and St2 components of Streptomyces thermoautotrophicus nitrogenase are shown in SEQ ID NOs: 5-8 (Genebank accession numbers: KF951061, KF951060, KF951059 and KF956113). Exemplary full length polypeptide sequences of St1 and St2 of Streptomyces thermoautotrophicus nitrogenase are shown in SEQ ID NOs: 21-24. Due to the degeneracy of the genetic code many alternate nucleic sequences to those specifically described herein can encode the nitrogenase subunits and other amino acid sequences discussed herein, and therefore those are encompassed by the present invention as well.
This invention encompasses sequence homologs of Streptomyces thermoautotrophicus nitrogenase components, which can be identified by computer data mining or sequence alignment techniques described in this specification and well known in the art. In addition, while St3 is the actual functional carbon monoxide dehydrogenase donor supplying components St1 and St2 of Streptomyces thermoautotrophicus nitrogenase (Ribbe et al, J Biol Chem, 1997, 272 (42):26627-33), component St1 itself bears a certain degree of homology to carbon monoxide dehydrogenase (CODH) types of enzymes (other names in common use include carbon-monoxide dehydrogenase, anaerobic carbon monoxide dehydrogenase, carbon monoxide oxygenase, and carbon-monoxide:(acceptor) oxidoreductase). Thus other homologs of CODH enzymes, which utilize N2 as a substrate instead or in addition to CO gas, are functional homologues of Streptomyces thermoautotrophicus nitrogenase. Thus this invention encompasses all such enzymes, being either sequence homologs of Streptomyces thermoautotrophicus nitrogenase or being CODH enzymes capable of fixing nitrogen. In addition, component St2 bears homology to reductase types of enzymes, including superoxide dismutases (SOD), which can be used in conjunction with St1 type of enzymes. Methods to identify homologs of these types of enzymes are well known in the art, and may include data mining techniques, sequence alignment techniques, identification of homologs with similar functional domains, etc.
This invention prospectively and potentially may become applicable to other nitrogenase systems. For instance, the free-living diazotrophic bacterium Klebsiella pneumoniae possess a nitrogenase complex encoded by a cluster of nif genes (Halbleib and Ludden, J Nutr, 2000, 130:1081-4). The three structural subunits of the nitrogenase are encoded by nifHDK genes, with other nif cluster genes involved in auxiliary metabolic functions. Exemplary nif gene sequences of Klebsiella pneumoniae and Azotobacter vinelandii sequences are shown in SEQ ID NOs: 9 and 10, respectively. Additional diazotrophs are found amongst Azotobacter, Rhizobium, certain cyanobacteria, as well as other species. The instant invention contemplates the use of any nitrogenase from any organism, similarly to the use of Streptomyces thermoautotrophicus nitrogenase, which potentially and prospectively can be used in its native or modified form (for instance via mutagenesis, directed evolution, codon optimization and other techniques known in the art) to create plants, plant cells or other heterologous cells and organisms capable of nitrogen fixation.
The nucleotide sequences of the nitrogenase encoding genes may be derived from wild-type organisms. Wild-type refers to the normal gene or organism found in nature without any known mutation. Other nucleotide sequences within the invention include nucleotide sequences that encode variants of the nitrogenase genes and proteins, and nucleotide sequences that encode mutant forms, recombinant forms, or non-naturally occurring variant forms of these genes and proteins, which exhibit about 50% to about 200%, or more, of the biological/enzymatic activity of the protein in question as determined by the assays known in the art.
Heterologous Cells, other than Plant Cells, Comprising Streptomyces thermoautotrophicus Nitrogenase
Similarly to heterologous expression in plants, Streptomyces thermoautotrophicus nitrogenase can be expressed in other types of organisms and cells, which are not naturally capable of nitrogen fixation, for either generation of novel organisms capable of nitrogen fixation, or for research, or for studies of the nitrogenases. In one particular aspect of the present invention, nitrogenase (or nitrogenase complex) from Streptomyces thermoautotrophicus can be expressed in a variety of organisms where the nitrogenase, or any nitrogenase activity, is not naturally found. Non-limiting examples of desirable unicellular and multicellular organisms for such modification include bacteria (other than Streptomyces thermoautotrophicus) belonging to eubacteria and archea; cyanobacteria; fungi, including yeast, mycorrhizae, molds or mushrooms; protists and algae; or animals. Organisms particularly preferable for Streptomyces thermoautotrophicus nitrogenase expression are certain bacteria, fungi and algae. Any organism or cell type where the specific nitrogenase is not naturally found can be used for heterologous nitrogenase expression, resulting in a novel trait of nitrogen fixation in the heterologous cell or organism.
Microorganisms expressing nitrogenase and producing biologically available nitrogen are particularly useful in agriculture, for instance as biofertilizers which can be applied to soil, seed or plant surfaces. Non-limiting examples of such organisms include rhizobia, mycorrhizal fungi, pink-pigmented facultative methylotrophs (PPFM bacteria) and plant-growth promoting and plant colonizing microorganisms, for example yeast, algae, bacteria, etc. and methods of use, as known in the art. In one embodiment, these organisms express heterologous nitrogenase from Streptomyces thermoautotrophicus. By way of example only, heterologous nitrogenase from Streptomyces thermoautotrophicus can be expressed in bacteria such as E. coli, for instance, for studies of the nitrogenase complex or for expression and purification of the nitrogenase proteins. In another embodiment, nitrogenases can be expressed in unicellular or multicellular algae, which can be used as biofertilizers or in the production of biofuels. Methods for construction of transformation vectors and stably or transiently transforming unicellular and multicellular organisms, including bacteria, fungi, algae or animals, are well known in the art and are not described here for brevity.
Genetically Modified Plants, Plant Cells and Heterologous Cells Capable of Nitrogen Fixation
The terms “transgenic,” “transformed,” and “transfected” as used herein include any cell, cell line, callus, tissue, plant tissue, plant or organism into which a nucleic acid heterologous to the host cell has been introduced. The term “transgenic” as used herein does not encompass an alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events, such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. The term “transgenic plant” refers to a plant or plant tissue that contains an inheritable heterologous nucleotide sequence. The present invention also encompasses progeny, whether produced sexually or asexually, or through breeding techniques, of plants covered by the present invention or containing sequences disclosed herein.
The term “plant” is used broadly herein to refer to a eukaryotic organism containing a plastid or plastids, and being at any stage of development. The term “plant” as used herein refers to a whole plant or a part of a plant (e.g., a plant cutting, a plant cell, a protoplast, a plant cell culture, a plant organ, a plant seed, and a plantlet), a seed, a cell- or a tissue-culture derived from a plant, plant organ (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, etc.), as well as unicellular or multicellular algae. Any plant may be used in this invention. This includes flowering and non-flowering plants, such as algae, monocots or dicots, and C3 and C4 plants. In one aspect for research and other purposes Arabidopsis thaliana or Nicotiana tabacum (tobacco) can be used as they are preferred model organisms in plant research. In another aspect, as marketable products and for agricultural or horticultural purposes, plants such as cotton, corn, wheat, various row crops, other food crop plants, ornamental, horticultural and other plants can be used.
Oilseed plants, e.g. plants that produce seeds or fruit with oil content from about 5% to about 50%, or more. Exemplary oilseed or oil crop plants useful in practicing the methods disclosed herein include, but are not limited to sunflower; sesame; soybean; mustard; coconut; cotton; peanut; rice; wheat; flax (linseed); sunflower; olive; corn; palm; palm kernel; sugarcane; castor bean; switchgrass, as well as, plants in the genera of Brassica (e.g., rapeseed/canola; Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea); Camelina; Jatropha (Simmondsia chinensis); Miscanthus; Borago officinalis; Ricinus communis; Coriandrum sativum; Echium plantagineum; Cuphea hookeriana; Cuphea plucherrima; Cuphea lanceolata; Crepis alpina; Crambe abyssinica; Vernonia galamensis and Momordica charanita. These include major and minor oil crops used, or being investigated and/or developed to be used as sources of biofuels due to their significant oil production and accumulation. The present invention also encompasses plants that may be used for production of biomass, for example, for biofuel production (ex. ethanol production from plant cell-wall constituents), which may include exemplary crops such as corn, soybeans, grasses, and other plants known in the art.
Agricultural plants, e.g. plants produced by agricultural practices for human food, animal feed and variety of plant products, are highly desirable targets for genetic modification with Streptomyces thermoautotrophicus nitrogenase. Examples of agricultural plants include, but not limited to, corn, cotton, soybeans, wheat, rice, tomatoes, potatoes, sugar cane, palms, beans, fruits and vegetables, sugar beet, sunflower and plethora of additional agricultural plants well known in the art.
The transgenic plant, heterologous cell or organism capable of nitrogen fixation, as used herein, includes at least one cell, i.e. one or more cells. In plants, a “plant cell” refers to any cell of a plant, either taken directly from a seed or plant, or derived through culture from a cell or a tissue taken from a plant. A “plant cell” includes, for example, cells from undifferentiated tissue (e.g., callus), plant seeds, propagules, gametophytes, sporophytes, pollen, microspores, embryos, etc.
The transgenic plant or heterologous cell capable of nitrogen fixation further includes an expressible heterologous nucleotide sequence. The term “expressible,” “expressed,” and variations thereof refer to the ability of a cell to transcribe a nucleotide sequence to mRNA and translate the mRNA to synthesize a peptide or a polypeptide that provides a biological or biochemical function. For purposes of the present invention, this function includes Streptomyces thermoautotrophicus nitrogenase that catalyzes nitrogen fixation.
“Streptomyces thermoautotrophicus nitrogenase”, as applied to the UBT1 nitrogenase, refers to the complex of St1 plus St2 and, optionally, plus St3 (
As used herein, “heterologous” refers to that which is foreign or non-native to a particular host, genome, gene or protein. Accordingly, a “heterologous nucleotide sequence” or “transgene” refers to a nucleotide sequence that originates from a species foreign to the host organism, or if the nucleotide sequence originates from the same species as the host, the nucleotide sequence is substantially modified from its native form in composition and/or genomic locus by deliberate genetic manipulation, which is present in the genome in a different location from which it is normally found, or which is found in a copy number in which it is not normally present. For instance, Streptomyces thermoautotrophicus nitrogenase is heterologous to any organism that is not Streptomyces thermoautotrophicus, such as plants, algae, other bacteria, animals, fungi, etc. Hence, “heterologous organism” or “heterologous cell”, for the purpose of expression or transformation with Streptomyces thermoautotrophicus nitrogenase, is any organism or cell which is not Streptomyces thermoautotrophicus. The term “nucleotide sequence” refers to a sequence of two or more nucleotides, such as RNA or DNA. A “heterologous protein” refers to a protein that is foreign or non-native to a host cell and is typically encoded by a heterologous nucleotide sequence.
Plants encompassed by the present invention include both monocots and dicots, C3 and C4 plants, agricultural, horticultural and ornamentals plants, oilseed plants, plants utilized for biomass, and algae. Non-limiting examples include plants such as corn, cotton, oil producing palms, various row crops, petunias, grasses and other plants.
Also encompassed by the present invention are parts of such plants including, for example, a protoplast, a cell, a tissue, an organ, a cutting, an explant, a reproductive tissue, a vegetative tissue and biomass. Such parts further include an inflorescence, a flower, a sepal, a petal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament, an anther, a male or female gametophyte, a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of said plant in culture, a tissue of said plant in culture, an organ of said plant in culture, a callus, a homogenate, propagation material, germplasm, cuttings, divisions and propagations.
The term “plant product” as used herein encompasses, but is not limited to, plants, plant parts, biomass and plant molecules that are customarily used for human food or animal feed. Furthermore, as used herein, plant products are not limited to edible products alone, but also include plants, plant parts, biomass and plant molecules such as, for example, pigments, fibers, cellulose, plant oils, lipids, fatty acids, sugars, medicinally active molecules, etc., that are useful in commercial products and processes such as lubricants, paints, pharmaceuticals, biofuels and other useful commercial products and applications known in the art.
The present invention also encompasses progeny, whether produced sexually or asexually, or through breeding techniques, of plants covered by the present invention or containing sequences disclosed herein. In regard to methods of propagating plants encompassed by the present invention, methods of propagation and reproduction of such plants are well known in the art, and include both sexual and asexual techniques. Asexual reproduction is the propagation of a plant to multiply the plant without the use of seeds to assure an exact genetic copy of the plant being reproduced. Any known method of asexual reproduction which renders a true genetic copy of the plant may be employed in the present invention. Acceptable modes of asexual reproduction include, but are not limited to, rooting cuttings, grafting, explants, budding, apomictic seeds, bulbs, divisions, slips, layering, rhizomes, runners, corms, tissue culture, nucellar embryos and any other conventional method of asexual propagation. All these and other methods of propagation and reproduction of plants are encompassed by the present invention.
In one aspect, plants capable of nitrogen fixation are rendered sterile and incapable of reproduction. Methods of introduction of sterility traits into plants are well known in the art and not detailed here for brevity (ex. Mitsuda et al, Plant Biotech J, 2006, 4:325-32).
Vectors
The term “vector” as used herein refers to a vehicle used for introduction of a nucleotide sequence into a host. A vector may be a plasmid, a cosmid, a phage, a transposon, a virus, or any other suitable vehicle. Preferably, the vector is a plasmid. A variety of vectors for transformation of eukaryotes and prokaryotes, plant, bacterial, cyanobacterial, archeal, protist, fungal, algal and animal cells are well known in the art. A vector may include operably linked regulatory sequences useful for expression of a gene product in a host, i.e. an expression vector, including but not limited to a promoter, a ribosomal binding site, a leader sequence, an intercistronic expression element (IEE), an internal ribosome entry site (IRES), an enhancer or a terminator sequences. When operably linked, such regulatory sequences perform their known and expected functions, facilitating gene or other nucleotide sequence expression. In one preferred embodiment, the vector is a vector for transforming a plastid as described below in one of the aspects of the invention.
In one embodiment, the heterologous nucleotide sequence or sequences can be placed in a single vector. For example, all Streptomyces thermoautotrophicus nitrogenase subunit genes (St3 being optional) can be placed in a single vector. In another embodiment heterologous nucleotide sequences, such as genes of Streptomyces thermoautotrophicus ntirogenase complex, can be placed separately in different vectors, which then can be used to transform a target cell. The heterologous nucleotide sequence can additionally include at least one gene encoding a cofactor for enhancing or modifying nitrogenase activity.
Vectors suitable for stable transformation of a plant cell are known in the art, and any suitable vector amongst the many known can be used to generate plants comprising Streptomyces thermoautotrophicus nitrogenase. Accordingly, the nitrogenase genes may be delivered into nuclear, or chloroplast (plastid), or mitochondrial genomes, or maintained as episomes. In one embodiment, for the transformation of nuclear host DNA, the vector is a binary vector (Lee and Gelvin, Plant Physiol, 2008, 146:325-332). A “binary vector” refers to a vector that includes a modified T-region from Ti plasmid, which allows replication in E. coli and in Agrobacterium cells, and usually includes selection marker genes. Examples of binary vectors are described later on. In another embodiment, the vector is a plastid or a chloroplast transformation vector (Lutz et al, Plant Physiol, 2007, 145:1201-1210, and Maliga, Trends Biotechnol, 2003, 21:20-28). Typically, a transgene in a chloroplast transformation vector is flanked by “homologous recombination sites,” which are DNA segments that are homologous to a region of the plastome. The “plastome” refers to the genome of a plastid. The homologous recombination sites enable site-specific integration of a transgene comprising expression cassette into the plastome by the process of homologous recombination. Chloroplast transformation vectors are described in detail later on.
Description of the aforementioned vectors is only exemplary and is not intended to limit the scope of the present invention. Other transformation vectors for transformation of plant, bacterial, fungal, algal or animal cells and methods for such transformations are well known in the art and for brevity are not described here in detail.
Promoters, Terminators and Other Genetic Elements
The heterologous nucleotide sequences or vectors described herein may include regulatory sequences useful for expression of a gene product in a host, such as a promoter, terminator or other genetic elements. The term “promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence. A promoter drives expression of an operably linked nucleotide sequence. The term “operably linked” as used herein refers to linkage of a promoter to a nucleotide sequence such that the promoter mediates (drives) transcription of the nucleotide sequence. A “coding sequence” refers to a nucleotide sequence that encodes a specific amino acid sequence. A promoter is typically located upstream (5′) to the coding sequence, while a terminator is typically located downstream (3′) to the coding sequence. A variety of promoters are known in the art and may be used to facilitate expression of a gene. Examples of suitable promoters include constitutive promoters, plant tissue-specific promoters, fungal promoters, algal promoters, bacterial promoters, animal cell promoters, plant development (developmental stage) specific promoters, inducible promoters, circadian rhythm promoters, viral promoters, male or female germline-specific promoters, flower-specific promoters, chloroplast promoters, as well as other promoters well known in the art.
A “constitutive” promoter refers to a promoter that causes a gene to be expressed in all cell types at all times. An example of a constitutive plastid promoter is the chloroplast rrn (16S rRNA gene) promoter (SEQ ID NO: 11); an example of nuclear genomic constitutive plant promoters include the Cauliflower Mosaic Virus (CaMV) 35S promoter (SEQ ID NO: 12), which confers constitutive, high-level expression in most plant cells. Further examples of suitable constitutive promoters include the Rubisco small subunit (SSU) promoter, leguminB promoter, TR dual promoter, ubiquitin promoter, and Super promoter. Different heterologous nucleotide sequences or vectors may contain different promoters to prevent gene silencing when several transgenes are expressed in the same cell. Use of specific suppressors, such as P19 suppressor, to prevent transgene silencing is also well known in the art. Preferred constitutive promoters are strong promoters.
An “inducible” promoter refers to a promoter that is regulated in response to a stress or a stimulus, or is induced by a specific factor. Examples of inducible promoters include tetracycline repressor system, lac repressor system, copper-inducible system, salicylate-inducible system (such as the PR1a system), and alcohol-inducible system. Further examples include inducible promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental stresses or stimuli. Such stresses or stimuli include heat (ex. tomato hsp70 promoter or hsp80 promoter), light, hormones (ex. abscisic acid), chemicals (ex. methyl jasmonate or salicylic acid), increased salinity, drought, pathogen (ex. promoter of the PRP1 gene), heavy metals (ex. heavy metal-inducible metallothionein I promoter and the promoter controlling expression of the tobacco gene cdiGRP) and wounds (ex. pinll promoter).
A “tissue-specific” promoter as used herein refers to a promoter that drives expression of an operably linked nucleotide sequence in a particular tissue. A tissue-specific promoter drives expression of a gene in one or more cell types in a specific organ (such as leaves, or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as seed storage cells or leaf parenchyma). Examples include Gentiana triflora promoter for chalcone synthase (NCBI accession AB005484), a seed-specific promoter (such as β-conglycinin, napin promoter and phaseolin) and mature leaves-specific promoter (such as SAG promoter from Arabidopsis). Promoters responsible to the circadian rhythm cycle can also be used in the heterologous nucleotide sequence or vector. Such promoters include the native ELF3 promoter and the promoter from the chlorophyll a/b binding protein (CAB2 promoter). Further, a “developmental stage” promoter as used herein refers to a promoter that drives expression of an operably linked nucleotide sequence at a particular developmental stage of a plant. Examples of developmental stage promoters are known in the art.
Use of promoters of different strengths permits modulation of the level of expression of Streptomyces thermoautotrophicus nitrogenase, allowing modification (i.e. increase or decrease) in the level of the nitrogenase and accompanying nitrogen fixation activity appropriate for various types of plants (and other heterologous host cells and organisms) and environmental conditions, as desired or necessary. Manipulation of Streptomyces thermoautotrophicus nitrogenase gene dosage can also be used, alone or in combination with different strength promoters, to modulate nitrogen fixation activity to a desired level.
The heterologous nucleotide sequence or vector may also include a terminator or other genetic regulatory sequences. Terminator, or transcriptional terminator, is typically a genetic sequence that marks the end of a gene or an operon and promotes transcriptional termination. Examples of terminators include the chloroplast psbA terminator (SEQ ID NO: 13) and the eukaryotic Cauliflower Mosaic Virus (CaMV) 35S terminator (SEQ ID NO: 14). Additional genetic regulatory sequences may include, but are not limited, to elements such as internal ribosome entry sites (IRES), enhancers, leaders, Shine-Dalgarno sequences, PPR binding sequences and intercistronic expression elements (IEE) (Zhou et al, Plant J, 2007, 52(5): 961-972), as well as other regulatory elements known in the art.
Markers
A vector may include a nucleotide sequence for a selectable and/or screenable marker. A “selection marker” refers to a protein necessary for survival or growth of a transformed plant cell grown in a selective culture regimen. Typical selection markers include sequences that encode proteins, which confer resistance to selective agents, such as antibiotics, herbicides or other toxins. Examples of selection markers include genes for conferring resistance to antibiotics, such as spectinomycin, streptomycin, tetracycline, ampicillin, kanamycin, G418, neomycin, bleomycin, hygromycin, methotrexate, dicamba, glufosinate, or glyphosate. Various other selection markers confer a growth-related advantage to the transformed cells over the non-transformed cells. Examples include selection markers for β-glucuronidase (in conjunction with, for example, cytokinin glucuronide), mannose-6-phosphate isomerase (in conjunction with mannose), and UDP-galactose 4-epimerase (in conjunction with, for example, galactose).
Selection markers include those which confer resistance to spectinomycin (e.g. encoded by the resistance gene aadA, SEQ ID NO: 15), streptomycin, kanamycin, lincomycin, gentamycin, hygromycin, methotrexate, bleomycin, phleomycin, blasticidin, sulfonamide, phosphinothricin, chlorsulfuron, bromoxynil, glyphosate, 2,4-D, atrazine, 4-methyltryptophan, nitrate, S-aminoethyl-L-cysteine, lysine/threonine, aminoethyl-cysteine or betaine aldehyde. Preferably, the selection marker is functional when expressed either from plant nuclear, plastid or mitochondrial genomes. Selection markers functional in the heterologous cells and organisms are also useful. Especially preferred are the genes aadA (GeneBank NC_009838), nptlI (GeneBank FM177583), BADH (GeneBank AY050316) and aphA-6 (GeneBank X07753).
After a heterologous nucleotide sequence has been introduced into a host cell, it may be advantageous to remove or delete certain sequences from the targeted genome. For example, it may be advantageous to remove the selection marker gene that has been introduced into a genome if the selection marker is no longer required after the selection phase is complete. Methods for directed deletion of sequences are known in the art. For example, the nucleotide sequence encoding a selection marker preferably includes a homology-based excision element, such as Cre-lox and attB/attP recognition sequences, which allow removal of the selection marker genes using site-specific recombinases (Lutz et al, Nat. Protoc., 2006, 1900-10).
In one embodiment, the heterologous nucleotide sequence or vector includes a reporter gene. Reporter genes encode readily quantifiable proteins which, via their color or enzyme activity, allow assessment of transformation efficiency, selection of the transformed cells, site or time of expression or identification of transformed cells. Examples of reporter genes include green fluorescent protein (GFP), luciferase, β-galactosidase, β-glucuronidase (GUS), R-Locus gene product, β-Lactamase, xy1E gene product, alpha-amylase and tyrosinase.
Functional Elements
The heterologous nucleotide sequence or vector may also include functional elements, which influence the generation, multiplication, function, use, expression and other parameters of the heterologous nucleotide sequence or vector used within the scope of the present invention. Examples of functional elements include replication origins (ORI), which make possible amplification of the heterologous nucleotide sequence or vector according to the invention in, for example, E. coli or in plant plastids; multiple cloning sites (MCSs), which permit and facilitate the insertion of one or more nucleic acid sequences; homologous recombination sites, allowing stable recombination of transgenes into plastid genome; border sequences, which make possible Agrobacterium-mediated transfer of the heterologous nucleotide sequence or vector into plant cells for the transfer and integration into the plant genome, such as, for example, the right or left border of the T-DNA or the vir region. The heterologous nucleotide sequence or vector may optionally include RNA processing signals, e.g. introns, which may be positioned upstream or downstream or within a polypeptide-encoding sequence in the heterologous nucleotide sequence. Intron sequences are known in the art to aid in the expression of heterologous nucleotide sequences in plant cells.
Targeting Sequences
In another embodiment, the heterologous nucleotide sequence includes a targeting sequence, such as a plastid targeting sequence. A “plastid targeting sequence” as used herein refers to a nucleotide sequence that encodes a polypeptide which can direct a second polypeptide to an organelle (ex. a plastid) in a cell. Preferably, plastid targeting sequence is a chloroplast targeting sequence. Mitochondrial and other organelle and compartment targeting sequences are also contemplated by the present invention.
It is known in the art that non-chloroplast proteins may be targeted to the chloroplast or other organelles by use of protein fusions with a peptide encoded by a targeting sequence. For example, nitrogenase genes may be fused with a plastid targeting sequence. When the nitrogenase gene is expressed, the targeting sequence is included in the translated polypeptide. The targeting sequence then directs the polypeptide into a plastid such as a chloroplast.
In one embodiment, the chloroplast targeting sequence is linked to a 5′- or a 3′-end of the nitrogenase genes. Typically, the chloroplast targeting sequence encodes a polypeptide extension (called a chloroplast transit peptide (CTP) or transit peptide (TP)). The polypeptide extension is typically linked to the N-terminus of the heterologous peptide encoded by the heterologous nucleotide sequence. Examples of a chloroplast targeting sequences include a sequence that encodes Nicotiana tabacum ribulose bisphosphate carboxylase (Rubisco) small subunit (RbcS) transit peptide, Arabidopsis thaliana EPSPS chloroplast transit peptide, Petunia hybrida EPSPS chloroplast transit peptide and rice rbcS chloroplast targeting sequence. Further examples of a chloroplast targeting peptide include the small subunit (SSU) of ribulose-1,5,-biphosphate carboxylase and the light harvesting complex protein I and protein II. Those skilled in the art will recognize that various chimeric constructs can be made, if needed, that utilize the functionality of a particular CTP to import a given gene product into a chloroplast. Other CTPs that may be useful in practicing the present invention include PsRbcS derived CTPs (Pisum sativum Rubisco small subunit CTP), AtRbcS CTP (Arabidopsis thaliana Rubisco small subunit 1A CTP), AtShkG CTP (CTP2), AtShkGZm CTP (CTP2synthetic; codon optimized for monocot expression), PhShkG CTP (Petunia hybrida EPSPS; CTP4; codon optimized for monocot expression), TaWaxy CTP (Triticum aestivum granule-bound starch synthase CTPsynthetic, codon optimized for corn expression), OsWaxy CTP (Oryza sativa starch synthase CTP), NtRbcS CTP (Nicotiana tabacum ribulose 1,5-bisphosphate carboxylase small subunit chloroplast transit peptide), ZmAS CTP (Zea mays anthranilate synthase alpha 2 subunit gene CTP) and RgAS CTP (Ruta graveolens anthranilate synthase CTP). Other transit peptides that may be useful include maize cab-m7 signal sequence and the pea (Pisum sativum) glutathione reductase signal sequence. Additional examples of such targeting sequences may include: spinach lumazine synthase, Chlamydomonas ferredoxin, and Rubisco activase transit peptides, and other sequences known in the art.
Variants
The present invention further relates to variants of the nucleotide sequences described herein. Variants may occur naturally, such as a natural allelic variant or variant from a related species. Other variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. These variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Preferably, the variant is a silent substitution, addition or deletion, which does not alter the properties and activities of the peptide encoded by the nucleotide sequence described herein. Conservative substitutions are also preferred.
Further embodiments of the invention include variant nucleotide or amino acid sequences comprising a sequence having at least 80% sequence identity or homology, and more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or homology, to the nucleotide or amino acid sequence of the nitrogenase or one of the nitrogenase functional domains or subunits. For example, a variant nucleotide sequence that is at least 95% sequence identical to a nitrogenase sequence is identical to the latter sequence except that the variant nucleotide sequence may include up to five point mutations per each 100 nucleotides of the nitrogenase sequence. In other words, to obtain a variant nucleotide sequence that is at least 95% identical to a nitrogenase nucleotide sequence, up to 5% of the nucleotides in the claimed sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides may be inserted into the nitrogenase sequence.
These mutations of the nitrogenase or nitrogenase functional domain sequences or subunits may occur at the 5′ or 3′ terminal positions of the sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the nitrogenase sequence or in one or more contiguous groups within the nitrogenase sequence. The term “sufficiently identical” as used herein refers to a first nucleotide sequence that contains a sufficient or minimum number of identical or equivalent nucleotides to a second nucleotide sequence, such that the first and second nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, nucleotide sequences that share common structural domains having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity across the sequences, and share a common functional activity are defined herein as sufficiently identical.
A “nitrogenase protein”, or “subunit thereof”, or any other protein or peptide presently disclosed and utilized in any of the methods and plants, or other organisms disclosed herein, refers to a protein or peptide exhibiting enzymatic or functional activity similar or identical to the enzymatic or functional activity of the specifically named protein or peptide. Enzymatic or functional activities of the nitrogenase proteins and peptides disclosed herein are described in Ribbe et al, J Biol Chem, 1997, 272(42):26627-33. “Similar” enzymatic/functional activity of a protein or peptide can be in the range from about 50% to about 200%, or more, of the enzymatic or functional activity of the specifically named protein or peptide when equal amounts of both proteins or peptides are assayed, tested or expressed as described in Ribbe et al., supra, or below under identical conditions and can therefore be satisfactory substituted for the specifically named proteins or peptides in present methods and transgenic plants, algae and other organisms encompassed herein to catalyze atmospheric nitrogen fixation.
To determine percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second nucleotide sequence for optimal alignment). For example, when aligning a first sequence to a second sequence having 10 nucleotides, at least 70%, preferably at least 80%, more preferably at least 90% of the 10 nucleotides between the first and second sequences are aligned. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, the length of the sequences, and the length of each gap that need to be introduced for optimal alignment of the two sequences. Computer software and algorithms known in the art may be used to determine percent identity between two given sequences.
As used herein, the phrase “sequence identity” means the percentage of identical nucleotide or amino acids residues at corresponding positions in two or more sequences when said sequences are aligned to maximize sequence matching, i.e. talking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in: Biocomputing: Informatics and Genome Projects, Smith D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje G., Academic Press, 1987; Computational Molecular Biology, Lesk A. M., ed., Oxford University Press, New York, 1988; Computer Analysis of Sequence Data, Part I, Griffin A. M. and Griffin H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis Primer, Gribskov M. and Devereux J., eds., M Stockton Press, New York, 1991; and Carillo H. and Lipman D., SIAM J. Applied Math., 48:1073 (1988). Methods to determine identity can also be found in publicly available computer programs.
Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, by the homology alignment algorithms, by search for similarity method or, by computerized implementations of these algorithms (BLAST, GAP, BESTFIT, PASTA and TEASTA in the GCG Wisconsin Package, available from Accelrys Inc., San Diego, Calif., USA), or other algorithms and methods known in the art. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is available in public access at NCBI/NIH and originally described in Altschul S. et al., NCBI NLM NIH Bethesda, Md., and Altschul S. F. et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publically available through the National Center for Biotechnology Information (NCBI).
Enhanced and/or Modified Nitrogenase Activity
In one aspect, nitrogen fixation levels can be increased or decreased by an increase or decrease in activity or level of enzymes involved in the nitrogen fixation reaction. For example, a nitrogenase can be expressed under a strong promoter, thereby allowing increase in concentration of the nitrogenase and nitrogenase-related proteins within a given cell and thus higher N2 fixation levels, as compared to a cell with a weaker promoter. Use of a strong constitutive promoter is expected to permit transformed plants to express high levels of the nitrogenase, and fix nitrogen, over prolonged periods of time. Use of inducible, tissue-specific and developmental stage promoters permits transformed plants to express the nitrogenase, and fix nitrogen, during development when and where in the plant specifically needed or desired.
Alternatively, nitrogenase levels, and concomitant nitrogen fixation, can be increased by expressing multiple copies of the enzyme complex, including expression from different organelles, such as plastids, nuclei, mitochondria, or from vector(s) maintained as an episome, or various combinations thereof, simultaneously. Once again, use of a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage promoter for such expression can be employed to controllably modulate nitrogen fixation to achieve desired levels as well as stage of development and location within plants.
Thus, nitrogenase levels and concomitant levels of N2 fixation can be controllably modulated to optimize plant or other host cell, or organism, growth by use of promoters of different strength, tissue specificity, inducibility, developmental stage specificity and duration of transcriptional activity. Nitrogenase levels and levels of N2 fixation can also be modulated by the extent of gene dosage (copy number) in any single intracellular genome, or combination of genomes. In addition to employing any of these strategies alone, combinations of any of the forgoing methods can be used to optimize N2 fixation compared to that in control or wild type plants or host cells or organisms, or in prototype versions of transgenic plants or host cells or organisms.
The term “enhance”, “enhanced”, “increase”, “increased”, “decrease”, “decreased”, “modify”, “modified”, “optimize”, “optimized”, “modulate” or “modulated”, and the like, refer to a statistically significant increase or decrease in a parameter or value herein. For the avoidance of doubt, these terms refer to about a 5% increase in a given parameter or value, about 10% increase, about a 15% increase, about a 20% increase, about a 25% increase, about a 30% increase, about a 35% increase, about a 40% increase, about a 45% increase, about a 50% increase, about a 55% increase, about a 60% increase, about a 65% increase, about a 70% increase, about a 75% increase, about an 80% increase, about an 85% increase, about a 90% increase, about a 95% increase, about a 100% increase, or more, over the baseline value, or comparative parameter value in a prototype trangenic organism, and similarly for decreases, modifications, optimizations, etc. These terms also encompass continuous ranges consisting of any lower indicated value or any higher indicated value, including ranges of any pints in between, for example, from about 5% to about 50%, etc. Any ranges that can be formed by any of the values or data presented herein represent further embodiments of the present invention.
In another aspect, use of a variety of translational, transcriptional and other enhancing elements (e.g., IEE sites, enhancers, etc.), as well as co-expression of additional proteins allowing to stabilize, enhance or improve nitrogenase activity, can be used to enhance or modify plant nitrogen fixation.
In yet another aspect, methods to modify and increase nitrogenase and/or other related enzymes activities may include directed evolution, codon optimization, protein engineering, rational design and other similar methods well known in the art. These and other methods are known to significantly improve enzyme activity, selectivity, stability and other parameters, as compared to an identical naturally occurring enzyme that has not undergone these improvement processes (ex. Cobb et al, Curr Opin Chem Biol, 2012, 16(3-4):285-91).
Cofactors
As used herein, the term “cofactor” refers to an organic molecule, an inorganic molecule, a peptide, a protein, or a nucleotide required for or enhancing an enzyme activity. Examples of co-factors useful for enhancing nitrogenase activity may include manganese, molybdenum or iron ions, polypeptides, CoxL, CoxM and CoxS proteins, and other molecules.
Nitrogenase Crosstalk
In one aspect, the invention relates to nitrogenase crosstalk, where a first heterologous sequence includes gene or genes coding for nitrogenase, wherein said first heterologous nucleotide sequence is operably linked to a first promoter. The nitrogenase crosstalk further comprises a vector having a second heterologous nucleotide sequence operably linked to a second promoter.
For example, the promoter for the first heterologous nucleotide sequence, the nitrogenase, is inducible by the second heterologous nucleotide sequence, a transcription factor. An exemplary inducible promoter is T7 promoter, which is activated by T7 RNA polymerase. Yet another exemplary inducible promoter is the UAS promoter, inducible by Gal4 binding domain fused to a VP16 transcriptional activator. There are other multiple examples of inducible promoter/activator pairs known in the art. In one embodiment, the promoter for the second heterologous nucleotide sequence is a tissue-specific promoter. The second heterologous nucleotide sequence, by way of example only, is a T7 RNA polymerase. Accordingly, when the tissue specific promoter is activated, the gene for the T7 RNA polymerase will be transcribed and activate the nitrogenase gene driven by the inducible T7 promoter. Thus, nitrogenase activation is indirect and occurs via nitrogenase crosstalk.
Methods for Producing Plants, Plant Cells and Heterologous Cells Comprising a Nitrogenase
In one aspect, the invention describes creation of genetically modified plants capable of fixing nitrogen on their own. Genetically modified plants capable of nitrogen fixation further include an expressible heterologous nucleotide sequence. The term “expressible,” “expressed,” and variations thereof refer to the ability of a cell to transcribe a nucleotide sequence to mRNA and translate the mRNA to synthesize a polypeptide that provides a biological or biochemical function. Preferably, the cell is a plant cell. As used herein, “heterologous” refers to that which is foreign or non-native to a particular host or genome. Accordingly, a “heterologous nucleotide sequence” or “transgene” refers to a nucleotide sequence that originates from a species foreign to the host organism, or if the nucleotide sequence originates from the same species as the host, the nucleotide sequence is substantially modified from its native form in composition and/or genomic locus by deliberate genetic manipulation, or is present in a location in a genome in which it is not normally found, or is present in more than the usual number of copies. The term “nucleotide sequence” refers to a sequence of two or more nucleotides, such as RNA or DNA. A “heterologous protein” refers to a protein that is foreign or non-native to a host cell and is typically encoded by a heterologous nucleotide sequence.
The term “transfecting” or “transforming” refers to introducing a nucleotide sequence into a host cell or into a plastid of the cell. The nucleotide sequence that is being introduced to the host cell nuclear genome, or plastid genome, or mitochondrial genome, or maintained as an episome, or other location within the cell, may include a heterologous nucleotide sequence or a vector as described above. Transfection of the heterologous nucleotide sequences is achieved by methods known to a skilled artisan. Any method that permits the introduction of a nucleotide sequence into a plant cell is suitable. Examples of such methods include transformation of chemically competent cells, microinjection, electroporation, biolistic bombardment with DNA-coated microparticles (“gene gun” method), permeabilizing a cell with polyethylene glycol, silicon whiskers, fusion with other DNA-comprising units such as minicells, hybridomas, hybrid cells or liposomes. Preferred methods include, for example, biolistic gene delivery and Agrobacterium mediated transformation.
Similarly, a variety of vectors and methods are known in the art to introduce heterologous sequences into bacterial (including cyanobacteria), fungal, algal or animal cells, thus resulting in transgenic organisms expressing the heterologous sequences. These methods are well known to a skilled artisan and can be used to stably or transiently express nitrogenase in a heterologous system. For the sake of brevity, only plant transformation methods will be described in detail and all other methods known in the art are incorporated herein by reference in their entirety.
Methods for regulating biological processes are known in the art, and may include various constitutive or inducible promoters, enhancers or silencing sequences and other means. These methods can be used to up- or down-regulate nitrogen fixation capacity of cells expressing Streptomyces thermoautotrophicus nitrogenase.
Plant Nuclear Genome Transformation
In one aspect, Agrobacterium is an effective tool for transforming plant nuclear genomes. The process of plant genetic transformation by Agrobacterium has been extensively characterized and is well known in the art. Agrobacterium T-DNA is used as a vehicle for delivering the gene or genes of interest (GOI) into the host genome. Initially, this technology was based on cloning GOIs directly into the T-DNA region of the Ti plasmid. However, this approach was technically challenging due to the large size and low copy number of Ti plasmids, leading to difficulties in plasmid isolation and manipulation. It was replaced by binary vector systems (Lee and Gelvin, Plant Physiol, 2008, 146:325-332) composed of two plasmids: (i) the helper plasmid—the Agrobacterium Ti plasmid carrying the vir genes, but lacking a functional T-DNA segment, and (ii) the binary vector constructed on a DNA backbone derived from commonly used E. coli cloning vectors and carrying the GOI flanked by 25 bp-long right and left T-DNA border sequences (RB and LB) (
Agrobacterium armed with an appropriate binary vector is used to generate transgenic plants. Arabidopsis thaliana and Nicotiana tabacum are among the most commonly used model organisms for both nuclear and chloroplast genetic transformation due to well-developed and efficient protocols for DNA delivery and recovery of transformants. Stable nuclear transformation of Arabidopsis germline cells can be rapidly achieved by the floral-dip method, where flowers are dipped in liquid Agrobacterium culture. Seeds from the dipped plants are collected and geminated on herbicide-supplemented selective media, which allows only transgenic plants expressing the selection marker gene contained in the T-DNA to survive. For tobacco, leaf disk inoculation, is a commonly used method to produce transgenic plants. Leaf disks are submerged in liquid Agrobacterium culture and then transferred to a callus inductive medium that has been supplemented with appropriate herbicide and plant hormones. The herbicide resistance gene contained in the T-DNA ensures that only the transformed cells survive, and the plant hormones promote regeneration of plants from the surviving cells. Another highly preferred method is plant transformation using biolistic DNA delivery, and additional methods of transformation by electroporation and polyethylene glycol (PEG)-mediated DNA delivery are known in the art. Any suitable known method known can be used to produce plants comprising a nitrogenase. Particularly preferred are commercial plants including corn, cotton, various row crops, ornamental plants, as well as any other agriculturally or commercially important plant.
Plastid Genetic Engineering
A plant cell typically contains a “plastid,” which refers to an organelle with its own genetic machinery in a plant cell. Examples of plastids include chloroplasts, chromoplasts, etioplasts, gerontoplasts, leucoplasts, proplastids, amyloplasts, elaioplasts, etc. The prokaryotic nature of chloroplast DNA integration and gene expression mechanisms necessitates chloroplast transformation vectors to be constructed differently than binary vectors. Unlike Agrobacterium-mediated genetic transformation, where T-DNA integrates randomly, chloroplasts support homologous recombination allowing targeted integration of the transgene. Therefore, in chloroplast transformation vectors, the gene or genes of interest (GOI) can be flanked by two sequences homologous to the selected integration site within the genome, also known as LTR and RTR (
Unlike monocistronically expressed GOIs integrated into the nuclear genome, polycistronic gene expression in chloroplasts allows the use of only one promoter and terminator sequence for multiple ORFs organized in an operon-like gene group. In this arrangement, each ORF requires a separate ribosome binding site (RBS) for translation initiation. Numerous bacterial and plastid RBSs (including Shine-Dalgarno [AGGAGG] sequence), are known in the art. The polycistronic nature of chloroplast gene expression also permits easy cloning of multiple transgenes—particularly those derived from an existing bacterial operon—into chloroplast transformation vectors and their integration into the chloroplast genome in a single transformation step. Alternatively, nitrogenase genes can be arranged as an artificial operon for expression.
In one embodiment, the preferred method of chloroplast transformation is biolistic DNA delivery. The biolistic chloroplast transformation methodology is well known in the art (Verma et al, Nat Prot, 2008, 3:739-758 and Lutz et al, Nat Prot, 2006, 1900-10). Briefly, tobacco leaf explants are bombarded with vector-coated gold microparticles and transplastomic plants are regenerated on a medium containing appropriate hormones and selection agents. Not all plant species can be transformed and regenerated using their leaf explants. Transplastomic plants of agronomically important species, such as cotton and soybean, are produced via somatic embryogenesis. Other methods for plastid transformation known in the art can also be used.
In one embodiment, nitrogenase genes from Streptomyces thermoautotrophicus can be expressed either as monocistronic mRNAs or as an operon (a polycistronic mRNA) from the plastidal genome, by integration via single or multiple vectors. The term “operon” refers to a nucleotide sequence which codes for a group of genes transcribed together. The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression. Some genes can be transcribed into mRNA and translated into polypeptides (structural genes); other genes can be transcribed into RNA (e.g. rRNA, tRNA), and other types of genes function as regulators of expression (regulator genes). Streptomyces thermoautotrophicus nitrogenase genes required for nitrogen fixation can be assembled in an operon and may include genes encoding for L, M and S subunits of component St1 (also referred as sdnL, sdnM and sdnS, respectively), and gene encoding for D subunit of component St2 (also referred as sdnO gene). The operon may optionally include, but is not limited to, genes CoxL, CoxM and CoxS of the component St3, or as required to restore a fully functional nitrogenase system within a specific plant cell.
The nitrogenase sequence (or nitrogenase subunits amino acid sequences) can alternatively be expressed from other nucleic sequences within a plant cell or a heterologous cell including nuclear or mitochondrial genomes (example of mitochondrial genome transformation can be found in Remade et al, PNAS USA, 2006, 103:4771-4776), episomes or other nucleic acid sequences (plasmid, viral, etc) which are known in the art.
The following presented examples are meant to be illustrative and not limiting of the practice or products of the present invention. The examples below show introduction of Streptomyces thermoautotrophicus nitrogenase genes into chloroplasts of a commonly known plant model organism, Nicotiana tabacum, as well as other plant species, and generation of plants comprising nitrogenase and capable of nitrogen fixation. Methods for generation of additional species of transgenic plants, or heterologous transgenic organisms, are well known in the art and are not described here in detail for conciseness.
An exemplary chloroplast transformation vector pCTV (Chloroplast Transformation Vector) for nitrogenase expression can be constructed on the basis of essentially any standard cloning vector. For example, a standard cloning vector pUC19 (GeneBank #L09137 and SEQ ID NO: 16) can be used. Any other suitable cloning vector can be used to construct a nitrogenase bearing vector. In this example, pUC19 multiple cloning site (MCS) was replaced by a sequence containing an expanded number of restriction enzyme recognition sites to allow cloning of multiple genetic elements. The new expanded MCS contained the following restriction sites: AgeI-AscI-SphI-BgIII-XhoI-EcoRI-SaclI-KpnI-EcoRV-NheI-SpeI-SalI-SacI-NdeI-BamHI-StuI-KasI-PacI-FseI-SwaI-HindIII-PstI/SbfI-NotI-SmaI. The chloroplast Prm promoter (SEQ ID NO: 11) was cloned as an AscI/SphI PCR fragment; other promoters, such as the chloroplast psbA promoter (GeneBank DQ463359, SEQ ID NO: 17), can be used. The chloroplast psbA terminator sequence (SEQ ID NO: 13) was cloned as a HindIII/PstI fragment; other suitable terminators known in the art can be used. The spectinomycin resistance gene aadA (SEQ ID NO: 15), driven by Shine-Dalgarno (AGGAGG or AGGAGGT) leader sequence was cloned into pCTV as a SphI/XhoI PCR fragment; other suitable selection markers are known in the art and can be used. To make a pCTV vector suitable for integration into the chloroplast genome, homologous recombination (HR) sequences were cloned to flank the nitrogenase expression cassette. In one embodiment, the integration site is the TrnI/TmA locus within tobacco chloroplast genome; other integration sites can be selected. The TrnI HR (SEQ ID NO: 18) was cloned as an AgeI/AscI PCR fragment, followed by TrnA HR (SEQ ID NO: 19) which was cloned as a PstI/NotI PCR fragment. An exemplary pCTV sequence and map are shown in SEQ ID NO: 20 and
In one embodiment, the nitrogenase compex from Streptomyces thermoautotrophicus is cloned into pCTV for expression in the form of synthetic operon containing genes: sdnL-sdnS-sdnM-sdnO, encoding for Streptomyces thermoautotrophicus nitrogenase proteins (SEQ ID NOs: 21-24). It should be noted, that genes in this operon can optionally be positioned in different order from that described herein. The cloning of the nitrogenase operon was performed, for technical simplicity, as two separate segments. The first segment comprising sdnL gene, optimized for expression in chloroplasts (designated as StNitF1; SEQ ID NO: 25), was cloned as a KpnI/NheI fragment. The second segment comprising sdnS-sdnM-sdnO genes, optimized for expression in chloroplasts (designated as StNitF2; SEQ ID NO: 26), was cloned as an NheI/NdeI fragment. Genes of the operon are driven by Shine-Dalgarno sequences and separated by intercistronic expression elements, resulting in pCTV-StNitrogenase vector suitable for chloroplast transformation (SEQ ID NO: 27 and
In addition to pCTV-StNitrogenase, a number of supplementary vectors have been prepared on the basis of pCTV. First, a vector for preparation of negative control N. tabacum plants have been generated by cloning a reporter beta-glucuronidase (GUS) gene downstream of aadA in pCTV vector. The resulting vector has been designated as pCTV-GUS and utilized to produce transplastomic plants that served as additional negative experimental controls, side by side with wild type tobacco plants. Yet another vector, carrying GUS positioned downstream of Streptomyces thermoautotrophicus nitrogenase in pCTV-StNitrogenase, vector has been constructed to serve as a supplementary experimental tool and was designated as pCTV-StNitrogenase-GUS. All results for plants generated using pCTV-StNitrogenase-GUS were similar to plants generated using pCTV-StNitrogenase and therefore are not detailed here for brevity.
Plants comprising Streptomyces thermoautotrophicus nitrogenase (experimental plants), as well as plants comprising GUS (control plants), were produced using methods well known in the art (Verma et al, Nat Prot, 2008, 3:739-758 and Lutz et al, Nat Prot, 2006, 1900-10). Briefly, 0.6 micron gold particles (BioRad) coated with the vector DNA were bombarded into leaves of aseptically grown 4-6 weeks old Nicotiana tabacum plants (cv. Petit Havana) using PDS-1000/He Biolistic Particle Delivery System (system settings: bombardment He pressure approx. 250 psi above rapture disk pressure, [rapture disks of 1,100 psi are typically used]; distance from the top of the chamber 9cm [third slot], chamber vacuum pressure 28 in Hg). The bombarded leaves were incubated at 25-26° C. in the dark for 2-3 days and dissected to 5×5 mm squares, which were placed in deep Petri dishes containing 50 ml of RMOP medium (RMOP per liter: MS salts [Caisson, according to manufacturer's instructions]; 100 mg myo-inositol; 1 mg thiamine HCl; 1 mg 6-benzylamino purine; 0.1 mg 1-naphthaleneacetic acid; 30 gr sucrose; 7-8 g phytoblend [Caisson], pH=5.8 adjusted with KOH, and supplemented with 500 μg/ml of spectinomycin [Sigma]). The Petri dishes were sealed with parafilm and cultivated under cool-white fluorescent lamps (1,900-2,000 lux) with 16 h light/8 h dark cycle at 26° C. Transgenic plants appeared within 4-8 weeks post bombardment. The plants were transferred and further aseptically maintained in magenta boxes on MSO medium (MSO per liter: MS salts [Caisson, according to manufacturer's instructions]; 30 gr sucrose; 7-8 g phytoblend [Caisson], pH=5.8 adjusted with KOH, supplemented with MS Vitamins [Phytotechnology Laboratories, according to manufacturer instructions]), and further grown under cool-white fluorescent lamps (1,900-2,000 lux) with 16 h light/8 h dark cycle at 26° C. Typically, pCTV-StNitrogenase plants regenerated after bombardment have shown chimeric/heteroplastomic phenotype (
All plants surviving spectinomycin selection regimen have been further validated using PCR and histochemical staining (for GUS expressing plants). Pairs of primers have been designed to specifically and accurately amplify DNA sequences integrated into the plant genome. Primer P1 (SEQ ID NO: 28), directed upstream from the TpsbA terminator, and primer P2 (SEQ ID NO: 29), directed downstream from Streptomyces thermoautotrophicus sequence, have been used to confirm nitrogenase comprising plants. Primers P1 and P3 (SEQ ID NO: 30), directed downstream from the GUS sequence, were used to identify GUS comprising plants. Both pairs of primers, P1+P2 and P1+P3, were designed to produce highly specific diagnostic bands of approx. 1 kb size when used to amplify pCTV-StNitrogenase and pCTV-GUS templates, respectively. DNA from leaves of the transformed aseptically grown plants was prepared using methods known in the art and used as a template in a PCR reaction driven by Taq polymerase (Takara); reaction products were resolved on 1% agarose gel. About half a dozen plants of each type have been positively identified, with exemplary results shown in
In addition, GUS carrying plants have been tested and confirmed for GUS expression using X-Gluc and methods well known in the art. Briefly, leaves or leaf parts from aseptically grown GUS expressing plants have been excised and incubated with 0.5 mg/ml of X-Gluc in phosphate buffer for 5-6 hours at 37° C., followed by overnight incubation with 75% EtOH solution for removal of chlorophyll.
Plants carrying Streptomyces thermoautotrophicus nitrogenase in their genome and produced as described in Examples 1 and 2 (“StNitrogenase plants”) showed phenotype highly resistant to nitrogen deficiency. Experimental plants, generated using either pCTV-StNitrogenase or pCTV-StNitrogenase-GUS vectors (both demonstrating similar results), have been compared to control plants, either wild type or plants generated using pCTV-GUS (both demonstrating similar results). Apical cuttings of experimental and control plants have been transferred to nitrogen deficient MSO medium (N-free MSO), comprising
per liter: N-free MS salts (MS Modified Basal Salt: w/o Nitrogen, Phytotechnology Laboratories, cat #M531, according to manufacturer's instructions); MS vitamins (Phytotechnology Labs); 30 gr sucrose; 7-8 g plant tissue culture agar (Sigma, A7921), pH=5.8 adjusted with KOH. Magenta boxes containing aseptically grown plants have been opened in a flow hood and aerated for approx. 5mins every 2-3 days to allow air exchange and atmospheric nitrogen access.
Within 7-10 days, control plants started showing clear signs of nitrogen deficiency, while experimental plants did not. First, typical symptoms of tobacco nitrogen deficiency in foliage started appearing in the control plants manifested as “fired” appearance of bottom leaves (Tucker, NCDA&CS, 1999, pp. 1-9), which started browning and curling at the tips, further spreading towards the leaf base. The experimental StNitrogenase plants, however, did not show these symptoms at this stage (
These results clearly demonstrate that plants comprising Streptomyces thermoautotrophicus nitrogenase exhibit strong resistance to nitrogen deficiency. While eventually, around three weeks after transfer to N-free MSO medium, the experimental plants also started to succumb and show nitrogen deficiency signs, they clearly showed a robust phenotypical resistance to nitrogen deficiency as compared to control wild type or GUS-only comprising plants. Strategies to further optimize and enhance nitrogenase expression and N2 fixation in transgenic plants are disclosed in “Promoters, terminators and other genetic elements”, “Enhanced and/or modified nitrogenase activity” and other sections above.
To investigate the mechanism behind nitrogen deficiency resistance of the experimental StNitrogenase plants, their capacity to fix atmospheric nitrogen was tested. Heavy nitrogen isotopes 15N (Sigma, Nitrogen-15N2, 98% atom, cat #364584) have been injected into magenta boxes containing aseptically grown experimental and control plants to a final concentration of 5% (vol/vol). Magenta boxes have been sealed air-tight and incubated for 6-7 days in standard growth conditions under cool-white fluorescent lamps with 16 h light/8 h dark cycle at 26-28° C. Upper plant parts have been collected, dried overnight at 50° C. and ground into a powder using mortar and pestle. To assess 15N enrichment levels, dried plant powder has been encapsulated (5 mg/sample) in tin capsules (COSTECH, cat #NC9464090) and sent for analysis to the Stable Isotope Facility at the University of California, Davis. The results, presented using standardized delta-15N values (Peterson and Fry, Ann Rev Ecol Syst, 1987, 18:293-320), demonstrated sizeable enrichment of approx. ˜20% in 15N content in experimental vs. control plants (average delta 15N of ˜297 vs. ˜367 for control and experimental plants, respectively,
Collectively, these results demonstrate that expression of Streptomyces thermoautotrophicus nitrogenase in plants enables them to use airborne N2 as a source of biologically available nitrogen, leading to strong resistance to nitrogen deficiency as compared to wild type or other control plants.
In the preceding examples we focused on generation and characterization of Nicotiana tabacum plants capable of nitrogen fixation. To demonstrate applicability of this technology to other plant species, we transformed additional plant species with Streptomyces thermoautotrophicus nitrogenase. Nicotiana sylvestris (cv. Only the Lonely) transformation has been conducted using constructs and methods described in Examples 1 and 2 above. Plants regenerating from post-bombardment callus have been excised and transferred to N-free MSO medium (as described in Example 3 above) side by side with wild-type N. sylvestris plants regenerated from leaf-derived callus grown on RMOP medium (as described in Example 2, without the antibiotics). As shown in
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure specifically described herein. Such equivalents are intended to be encompassed within the scope of the following claims.
This application claims benefit under 35 USC §119(e) of U.S. Provisional Application No. 61/991,103 filed on May 9, 2014, U.S. Provisional Application No. 62/008,597, filed on Jun. 6, 2014, and U.S. Provisional Application No. 62/091,046, filed on Dec. 12, 2014, the contents of each are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/029044 | 5/4/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62091046 | Dec 2014 | US | |
| 62008597 | Jun 2014 | US | |
| 61991103 | May 2014 | US |
