Patent Application
20200270616
The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1, <400>2, etc. The Sequence Listing, in written computer readable format (CRF), is incorporated by reference in its entirety.
Steviol glycosides are secondary metabolites extracted from the plants Stevia rebaudiana, Stevia phlebophylla, and Rubus chingii. These compounds have been used as non-caloric sweeteners that may be useful in preventing or reducing the prevalence and/or effects of diabetes. Terpenoid or isoprenoid pathways are the main source of steviol glycoside biosynthesis. Various attempts have been made to increase the yield and production of steviol glycosides, including the employment of agronomic practices, plant tissue culture, and microbial expression, but results thus far have not been optimized to correct for the naturally low yields of most secondary metabolites. Furthermore, one of the biggest challenges for production of a high quality stevia-based sweetener incorporating steviol glycosides is the elimination of the bitter aftertaste compared to conventional sucrose-based table sugar.
Common steviol glycosides include stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, dulcoside A. Although these compounds are available in some crops such as, for example, Stevia rebaudiana, it would be desirable to have a new method of producing steviol glycosides on an abbreviated time scale, to generate large quantities of these compounds to be added to food, pharmaceuticals, and nutritional supplements. The new method would, ideally, be inexpensive, would not result in the production of genetically-modified plants, would require fewer organic solvents for extraction than traditional methods, and would result in higher steviol glycoside production using less biomass than traditional methods, thus not requiring agronomic practices and large growing fields. Furthermore, the method would lead to the production of a sweetener without a bitter aftertaste.
Described herein are devices and methods for increasing the production of steviol glycosides, which have industrial and economic value. The steviol glycosides produced by the devices and methods disclosed herein do not require the ultra purification that is common in conventional or commercial methods and do not have a bitter aftertaste, making them better suited as flavor-enhancing additives to food, pharmaceutical, and nutritional supplement products.
The advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a plasmid” includes mixtures of two or more such plasmids, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase “optionally includes a reporter protein” means that the reporter protein may or may not be present.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint without affecting the desired result.
Throughout this specification, unless the context dictates otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer, step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, step, or group of elements, integers, or steps.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of any such list should be construed as a de facto equivalent of any other member of the same list based solely on its presentation in a common group, without indications to the contrary.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range was explicitly recited. As an example, a numerical range of “about 1” to “about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also to include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4, the sub ranges such as from 1-3, from 2-4, from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. The same principle applies to ranges reciting only one numerical value as a minimum or maximum. Furthermore such an interpretation should apply regardless of the breadth or range of the characters being described.
Disclosed are materials and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed compositions and methods. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed that while specific reference of each various individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a bacterium is disclosed and discussed and a number of different compatible bacterial plasmids are discussed, each and every combination and permutation of bacterium and bacterial plasmid that is possible is specifically contemplated unless specifically indicated to the contrary. For example, if a class of molecules, A, B, and C are disclosed as well as a class of molecules D, E, and F, and an example of a combination molecule, A-D, is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E is specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denote the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
DNA constructs are provided herein for the production of steviol glycosides. It is understood that one way to define the variants and derivatives of the genetic components and DNA constructs described herein is in terms of homology/identity to specific known sequences. Those of skill in the art readily understand how to determine the homology of two nucleic acids. For example, the homology can be calculated after aligning two sequences so that the homology is at its highest level. Another way of calculating homology can be performed according to published algorithms (see Zuker, M., Science, 244:48-52, 1989; Jaeger et al., Proc. Natl. Acad. Sci. USA, 86:7706-7710, 1989; Jaeger et al., Methods Enzyol. 183:281-306, 1989, which are herein incorporated by reference for at least material related to nucleic acid alignment).
As used herein, “conservative” mutations are mutations that result in an amino acid change in the protein produced from a sequence of DNA. When a conservative mutation occurs, the new amino acid has similar properties as the wild type amino acid and generally does not drastically change the function or folding of the protein (e.g., switching isoleucine for valine is a conservative mutation since both are small, branched, hydrophobic amino acids). “Silent mutations,” meanwhile, change the nucleic acid sequence of a gene encoding a protein but do not change the amino acid sequence of the protein.
It is understood that the description of mutations and homology can be combined together in any combination, such as embodiments that have at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology to a particular sequence wherein the variants are conservative or silent mutations. It is understood that any of the sequences described herein can be a variant or derivative having the homology values listed above.
In one aspect, a database such as, for example, GenBank, can be used to determine the sequences of genes and/or regulatory regions of interest, the species from which these elements originate, and related homologous sequences.
In one aspect, provided herein is a DNA construct having the following genetic components:
In one aspect, provided herein is a DNA construct having the following genetic components:
In another aspect, provided herein is a DNA construct having the following genetic components:
In still another aspect, provided herein is a DNA construct having the following genetic components:
In a further aspect, provided herein is a DNA construct having the following genetic components:
In yet another aspect, provided herein is a DNA construct having the following genetic components:
Each component of the DNA construct is described in detail below.
In one aspect, the nucleic acids described herein (e.g., genes that express HDR, hexokinase, a heat shock protein, GGPS, DXS, mevalonate-5-kinase, isopentenyl pyrophosphate isomerase, mevalonate pyrophosphate decarboxylase, a UDP glycosyltransferase, and/or an O-linked acetylglucosamine transferase) used in the DNA constructs described herein can be amplified using polymerase chain reaction (PCR) prior to being ligated into a plasmid or other vector. Typically, PCR-amplification techniques make use of primers, or short, chemically-synthesized oligonucleotides are complementary to regions on each respective strand flanking the DNA or nucleotide sequence to be amplified. A person having ordinary skill in the art will be able to design or choose primers based on the desired experimental conditions. In general, primers should be designed to provide for both efficient and faithful replication of the target nucleic acids. Two primers are required for the amplification of each gene, one for the sense strand (that is, the strand containing the gene of interest) and one for the antisense strand (that is, the strand complementary to the gene of interest). Pairs of primers should have similar melting temperatures that are close to the PCR reaction's annealing temperature. In order to facilitate the PCR reaction, the following features should be avoided in primers: mononucleotide repeats, complementarity with other primers in the mixture, self-complementarity, and internal hairpins and/or loops. Methods of primer design are known in the art; additionally, computer programs exist that can assist the skilled practitioner with primer design. Primers can optionally incorporate restriction enzyme recognition sites at their 5′ ends to assist in later ligation into plasmids or other vectors.
PCR can be carried out using purified DNA, unpurified DNA that is integrated into a vector, or unpurified genomic DNA. The process for amplifying target DNA using PCR consists of introducing an excess of two primers having the characteristics described above to a mixture containing the sequence to be amplified, followed by a series of thermal cycles in the presence of a heat-tolerant or thermophilic DNA polymerase, such as, for example, any of Taq, Pfu, Pwo, Tfl, rTth, Tli, or Tma polymerases. A PCR “cycle” involves denaturation of the DNA through heating, followed by annealing of the primers to the target DNA, followed by extension of the primers using the thermophilic DNA polymerase and a supply of deoxynucleotide triphosphates (i.e., dCTP, dATP, dGTP, and TTP), along with buffers, salts, and other reagents as needed. In one aspect, the DNA segments created by primer extension during the PCR process can serve as templates for additional PCR cycles. Many PCR cycles can be performed to generate a large concentration of target DNA or gene. PCR can optionally be performed in a device or machine with programmable temperature cycles for denaturation, annealing, and extension steps. Further, PCR can be performed on multiple genes simultaneously in the same reaction vessel or microcentrifuge tube since the primers chosen will be specific to selected genes. PCR products can be purified by techniques known in the art such as, for example, gel electrophoresis followed by extraction from the gel using commercial kits and reagents.
In a further aspect, the plasmid can include an origin of replication, allowing it to use the host cell's replication machinery to create copies of itself.
As used herein, “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of another. For example, if sequences for multiple genes are inserted into a single plasmid, their expression may be operably linked. Alternatively, a promoter is said to be operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence.
As used herein, “expression” refers to transcription and/or accumulation of an mRNA derived from a gene or DNA fragment. Expression may also be used to refer to translation of mRNA into a peptide, polypeptide, or protein.
In one aspect, the gene that expresses 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is isolated from plants such as, for example, plants from the mustard family. In one aspect, the mustard family plant includes Arabidopsis thaliana, Arabidopsis lyrata, canola or rapeseed, bok Choy, napa cabbage, rapini, turnip, cabbage, Savoy cabbage, red cabbage, collard greens, kale or ornamental kale, Brussels sprouts, kohlrabi, broccoli, cauliflower, or broccolini. In another aspect, the plant is wild flax or pink shepherd's purse. In a further aspect, the gene that expresses HDR has SEQ ID NO. 1 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses HDR is isolated from Arabidopsis thaliana and can be found in GenBank with accession number NM 119600.4.
Other sequences expressing HDR or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 1.
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis lyrata
Camelina sativa
Capsella rubella
Camelina sativa
Arabidopsis thaliana
Rhinolophus sinicus
Eutrema salsugineum
Thellungiela halophila
Lepidium apetalum
Raphanus sativus
Brassica napus
Brassica oleracea
Brassica napus
Brassica oleracea
Brassica rapa
Raphanus sativus
Brassica rapa
Brassica rapa
Brassica napus
Brassica napus
Tarenaya hassleriana
Tarenaya
hassleriana
Arabidopsis thaliana
Raphanus sativus
Brassica rapa
Brassica rapa
Juglans regia
Juglans regia
Populus euphratica
Populus trichocarpa
Populus trichocarpa
Lonicera hypoglauca
Lonicera dasystyla
Lonicera japonica
Camelina sativa
Erythranthe guttatus
Erythranthe guttatus
Oncidium hybrid cultivar
Isodon rubescens
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Phalaenopsis equestris
Phalaenopsis equestris
Capsella rubella
Raphanus sativus
Phalaenopsis equestris
Camelina sativa
Arabis alpina
Brassica rapa
Brassica napus
Brassica napus
Raphanus sativus
Raphanus sativus
Brassica oleracea
Brassica oleracea
Brassica oleracea
Brassica oleracea
Calothrix sp. 336/3
Oncidium hybrid cultivar
In one aspect, the gene that expresses hexokinase is isolated from a microorganism. In a further aspect, the microorganism is a fungus. In one aspect, the fungus is a yeast such as, for example, Saccharomyces cerevisiae. In a further aspect, the gene that expresses hexokinase has SEQ ID NO. 2 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect the gene that expresses hexokinase is isolated from Saccharomyces cerevisiae and can be found in GenBank with GI number M14410.1.
Other sequences expressing hexokinase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 2.
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
In one aspect, the gene that expresses a heat shock protein is isolated from a microorganism. In a further aspect, the microorganism is a fungus such as, for example, yeast. In a still further aspect, the yeast is Saccharomyces cerevisiae. In a further aspect, the gene that expresses a heat shock protein has SEQ ID NO. 3 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses a heat shock protein is isolated from Saccharomyces cerevisiae and can be found in GenBank with GI number X13713.
Other sequences expressing a heat shock protein or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 3.
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
In one aspect, the gene that expresses a geranylgeranyl pyrophosphate synthase (GGPS) is isolated from plants such as, for example, plants from the mustard family. In one aspect, the mustard family plant includes Arabidopsis thaliana, Arabidopsis lyrata, canola or rapeseed, bok choy, napa cabbage, rapini, turnip, cabbage, Savoy cabbage, red cabbage, collard greens, kale or ornamental kale, Brussels sprouts, kohlrabi, broccoli, cauliflower, or broccolini. In a further aspect, the gene that expresses GGPS has SEQ ID NO. 4 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses GGPS is isolated from Arabidopsis thaliana and can be found in GenBank with GI number NM 127943.3.
Other sequences expressing GGPS or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 4.
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
lyrata
Eutrema
salsugineum
Capsella
rubella
Arabis
alpina
Camelina
sativa
Camelina
sativa
Camelina
sativa
Brassica
oleracea
Brassica
napus
Brassica
napus
Brassica
napus
Brassica
rapa
Brassica
napus
Brassica
napus
Raphanus
sativus
Brassica
oleracea
Brassica
rapa
Raphanus
sativus
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Eutrema
salsugineum
Eutrema
salsugineum
Arabidopsis
lyrata
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Leucosceptrum
canum
Leucosceptrum
canum
Vigna
radiata
Sesamum
indicum
Jatropha
curcas
Populus
trichocarpa
Populus
euphratica
In one aspect, the gene that expresses steviol synthase or 1-deoxy-
Other sequences expressing steviol synthase or DXS or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 5.
Stevia
rebaudiana
Stevia
rebaudiana
Stevia
rebaudiana
Taraxacum
kok-saghyz
Ricinus
communis
Lycopersicon
hirsutum
Ipomoea
nil
Ipomoea
nil
Solanum
lycopersicum
Solanum
pennellii
Stevia
rebaudiana
Solanum
tuberosum
Tripterygium
wilfordii
Populus
euphratica
Gossypium
hirsutum
Gossypium
arboreum
Populys
trichocarpa
Gossypium
hirsutum
Theobroma
cacao
Herrania
umbratica
Herrania
umbratica
Theobroma
cacao
Solanum
pennellii
Solanum
lycopersicum
Solanum
lycopersicum
DXP synthase, or 1-deoxy-
In one aspect, the steviol glycosides are produced herein using starting materials from the non-mevalonate pathway for isoprenoid biosynthesis. In an alternative aspect, the starting materials for steviol glycoside biosynthesis used herein are generated via the mevalonate pathway. In still another aspect, the starting materials for steviol glycoside biosynthesis are generated from both the mevalonate and non-mevalonate pathways.
In one aspect, the gene that expresses mevalonate-5-kinase is isolated from a microorganism. In another aspect, the microorganism is a fungus such as, for example, a yeast. In still another aspect, the yeast is Saccharomyces cerevisiae. In a further aspect, the gene that expresses mevalonate-5-kinase has SEQ ID NO. 6 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses mevalonate-5-kinase is isolated from Saccharomyces cerevisiae and can be found in GenBank with accession number NC_001145.3.
Other sequences expressing mevalonate-5-kinase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 6.
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
In one aspect, the gene that expresses isopentenyl pyrophosphate isomerase is isolated from bacteria. In another aspect, the bacteria are from the genus Bacillus. In still another aspect, the bacteria are B. thuringiensis, B. anthraces, or B. cereus. In a further aspect, the gene that expresses isopentenyl pyrophosphate isomerase has SEQ ID NO. 7 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses isopentenyl pyrophosphate isomerase is isolated from Bacillus thuringiensis serovar konkukian str. 97-27 and can be found in GenBank with GI number NC_005957.1.
Other sequences expressing isopentenyl pyrophosphate isomerase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 7.
Bacillus
thuringiensis
Bacillus
thuringiensis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
anthracis
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
thuringiensis
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus sp. ABP14
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
cereus
Bacillus
cereus
Bacillus
thuringiensis
Bacillus
thuringiensis
Bacillus
thuringiensis
Bacillus
thuringiensis
In one aspect, the gene that expresses mevalonate pyrophosphate decarboxylase is isolated from bacteria. In another aspect, the bacteria are from the genus Lactobacillus. In still another aspect, the bacteria are L. paracasei or L. casei. In a further aspect, the gene that expresses mevalonate pyrophosphate decarboxylase has SEQ ID NO. 8 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses mevalonate pyrophosphate decarboxylase is isolated from Lactobacillus paracasei and can be found in GenBank with accession number NC_008526.1.
Other sequences expressing mevalonate pyrophosphate decarboxylase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 8.
Lactobacillus
paracasei
Lactobacillus
paracasei
Lactobacillus
paracasei
Lactobacillus
casei
Lactobacillus
paracasei
Lactobacillus
paracasei
Lactobacillus
paracasei
Lactobacillus
casei
Lactobacillus
casei
Lactobacillus
paracasei
Lactobacillus
casei
Lactobacillus
casei
Lactobacillus
casei
Lactobacillus
casei
Lactobacillus
paracasei
In one aspect, the gene that expresses UDP-glycosyltransferase is isolated from a plant. In another aspect, the plant is Stevia rebaudiana, sunflower (Helianthus annuus), soy (Glycine max), lettuce (Latuca sativa), Gossypia arboreum, Gossypium hirsutum, Gossypium raimondii, durian (Durio zibethinus), orange (Citrus sinensis), Clementine (Citrus clementina), pigeon pea (Cajanus cajan), grape (Vitis vinifera), carrot (Daucus carota), jujube (Ziziphus jujube), the common bean (Phaseolus vulgaris), or another common plant. In a further aspect, the gene that expresses UDP-glycosyltransferase has SEQ ID NO. 12 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. In one aspect, the gene that expresses UDP-glycosyltransferase is a UGT76G1 gene. In another aspect, the gene that expresses UDP-glycosyltransferase has one or more point mutations such as, for example, UGT76G1His155Leu. In certain aspects, the constructs described herein do not include a gene that expresses UDP-glycosyltransferase.
Other sequences expressing UDP-glycosyltransferase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 9.
Stevia rebaudiana
Stevia rebaudiana
Stevia rebaudiana
Stevia rebaudiana
Helianthus annuus
Helianthus annuus
Helianthus annuus
Helianthus annuus
Helianthus annuus
Helianthus annuus
Helianthus annuus
Latuca sativa
Helianthus annuus
Helianthus annuus
Helianthus annuus
Latuca sativa
Helianthus annuus
Helianthus annuus
Glycine max
Gossypium
arboreum
Gossypium
hirsutum
Gossypium
raimondii
Durio zibethinus
Gossypium
arboreum
Citrus sinensis
Citrus clementina
Gossypium
raimondii
Gossypium
raimondii
Helianthus annuus
Helianthus annuus
Helianthus annuus
Cajanus cajan
Gossypium
hirsutum
Gossypium
hirsutum
Gossypium
hirsutum
Gossypium
hirsutum
Populus
trichocarpa
Gossypium
hirsutum
Gossypium
hirsutum
Hevea brasiliensis
Gossypium
arboreum
Gossypium
arboreum
Gossypium
hirsutum
Gossypium
raimondii
Helianthus annuus
Helianthus annuus
Helianthus annuus
Gossypium
hirsutum
Durio zibethinus
Durio zibethinus
Vitis vinifera
Vitis vinifera
Daucus carota
Ziziphus jujube
Gossypium
hirsutum
Gossypium
hirsutum
Gossypium
hirsutum
Gossypium
hirsutum
Gossypium
raimondii
Gossypium
raimondii
Gossypium
raimondii
Gossypium
raimondii
Phaseolus vulgaris
Lobelia erinus
Lobelia erinus
Lobelia erinus
Gossypium
hirsutum
Quercus suber
Arachis ipaensis
Arachis ipaensis
Arachis ipaensis
Theobroma cacao
Theobroma cacao
Gossypium
hirsutum
Populus
euphratica
Populus
euphratica
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Quercus suber
Daucus carota
Vigna radiata
Populus
euphratica
Lobelia erinus
Latuca sativa
Helianthus annuus
Helianthus annuus
Manihot esculenta
Manihot esculenta
Jatropha curcas
Latuca sativa
Latuca sativa
Prunus avium
Gossypium
raimondii
Populus
euphratica
Lobelia erinus
Lobelia erinus
In one aspect, the gene that expresses O-linked acetylglucosamine transferase (OGT) is isolated from an animal. In another aspect, the animal is a mammal such as, for example, the thirteen-lined ground squirrel, domestic cow, domestic sheep, domestic cat or dog, water buffalo, domestic yak, olive baboon, chimpanzee, human, bonobo, gorilla, polar bear, sooty mangabey, drill, giant panda, Angola colobus, crab-eating macaque, goat, rhesus macaque, Southern pig-tailed macaque, the domestic horse, or another mammal. In a further aspect, the gene that expresses O-linked acetylglucosmamine transferase has SEQ ID NO. 13 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto.
Other sequences expressing O-linked acetylglucosamine transferase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 10.
Ictidomys
tridecemilineatus
Ictidomys
tridecemilineatus
Ictidomys
tridecemilineatus
Bubalus bubalis
Bos mutus
Bubalus bubalis
Bos mutus
Papio anubis
Bos taurus
Ursus maritimus
Papio Anubis
Ursus maritimus
Bos taurus
Bos taurus
Cercocebus atys
Cercocebus atys
Mandrillus
leucophaeus
Mandrillus
leucophaeus
Ailuropoda
melanoleuca
Macaca
fascicularis
Colobus angolensis
Ailuropoda
melanoleuca
Macaca
fascicularis
Capra hircus
Macaca mulatta
Ovis aries
Ovis aries
Macaca nemestrina
Colobus angolensis
Rhinopithecus
roxellana
Capra hircus
Marmota marmota
Macaca mulatta
Ovis aries
Ovis aries
Macaca nemestrina
Equus caballus
Canis lupus
familiaris
Rhinopithecus
roxellana
Chlorocebus
sabaeus
Canis lupus
familiaris
Chlorocebus
sabaeus
Equus caballus
Felis catus
Odocoileus
virgianus
Odocoileus
virgianus
Panthera pardus
Oryctolagus
cuniculus
Equus caballus
Fells catus
Odocoileus
virgianus
Odocoileus
virgianus
Panthera pardus
Oryctolagus
cuniculus
Pongo abelii
Gorilla gorilla
Panthera tigris
Callithrix jacchus
Leptonychotes
weddellii
Pantholops
hodgsonii
Pongo abelii
Gorilla gorilla
Callithrix jacchus
Panthera tigris
Leptonychotes
weddellii
Pan troglodytes
Equus asinus
Pan paniscus
Homo sapiens
Piliocolobus
tephrosceles
Pan troglodytes
Pan paniscus
Piliocolobus
tephrosceles
Equus asinus
Orycteropus afer
Homo sapiens
Pongo abelii
Homo sapiens
Homo sapiens
Equus asinus
Orycteropus afer
Homo sapiens
Homo sapiens
Delphinapterus
leucas
Lonchura striata
Ceratotherium
simum
Ceratotherium
simum
Microcebus
murinus
Saimiri boliviensis
Eptesicus fuscus
Peromyscus
maniculatus
Peromyscus
maniculatus
Delphinapterus
leucas
Lonchura striata
Ceratotherium
simum
Microcebus
murinus
Saimiri boliviensis
Homo sapiens
In another aspect, the DNA construct has the following genetic components: a) a gene that expresses 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, b) a gene that expresses hexokinase, c) a gene that expresses a heat shock protein, d) a gene that expresses a geranylgeranyl pyrophosphate synthase, and e) a gene that expresses 1-deoxy-
In an alternative aspect, the DNA construct has the following genetic components: a) a gene that expresses mevalonate-5-kinase, b) a gene that expresses isopentenyl pyrophosphate isomerase, c) a gene that expresses mevalonate pyrophosphate decarboxylase, d) a gene that expresses hexokinase, e) a gene that expresses a heat shock protein, f) a gene that expresses a geranylgeranyl pyrophosphate synthase, and g) a gene that expresses 1-deoxy-
In another aspect, the DNA construct has the following genetic components: a) a gene that expresses mevalonate-5-kinase, b) a gene that expresses isopentenyl pyrophosphate isomerase, c) a gene that expresses mevalonate pyrophosphate decarboxylase, d) a gene that expresses hexokinase, e) a gene that expresses a heat shock protein, f) a gene that expresses a geranylgeranyl pyrophosphate synthase, g) a gene that expresses a UDP glycosyltransferase, and h) a gene that expresses 1-deoxy-D-xylulose-5-phosphate synthase.
In yet another aspect, the DNA construct has the following genetic components: a) a gene that expresses mevalonate-5-kinase, b) a gene that expresses isopentenyl pyrophosphate isomerase, c) a gene that expresses mevalonate pyrophosphate decarboxylase, d) a gene that expresses hexokinase, e) a gene that expresses a heat shock protein, f) a gene that expresses a geranylgeranyl pyrophosphate synthase, g) a gene that expresses an O-linked acetylglucosamine transferase, and h) a gene that expresses 1-deoxy-D-xylulose-5-phosphate synthase.
In still another aspect, the DNA construct has the following genetic components: a) a gene that expresses mevalonate-5-kinase, b) a gene that expresses isopentenyl pyrophosphate isomerase, c) a gene that expresses mevalonate pyrophosphate decarboxylase, d) a gene that expresses hexokinase, e) a gene that expresses an O-linked acetylglucosamine transferase, f) a gene that expresses a geranylgeranyl pyrophosphate synthase, g) a gene that expresses and UDP glycosyltransferase, and h) a gene that expresses 1-deoxy-D-xylulose-5-phosphate synthase.
In another aspect, said construct further includes a) a promoter, b) a terminator or stop sequence, c) a gene that confers resistance to an antibiotic (a “selective marker”), d) a reporter protein, or a combination thereof.
In one aspect, the construct includes a regulatory sequence. In a further aspect, the regulatory sequence is already incorporated into a vector such as, for example, a plasmid, prior to genetic manipulation of the vector. In another aspect, the regulatory sequence can be incorporated into the vector through the use of restriction enzymes or any other technique known in the art.
In one aspect, the regulatory sequence is a promoter. The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence. In another aspect, the coding sequence to be controlled is located 3′ to the promoter. In another aspect, the promoter is derived from a native gene. In an alternative aspect, the promoter is composed of multiple elements derived from different genes and/or promoters. A promoter can be assembled from elements found in nature, from artificial and/or synthetic elements, or from a combination thereof. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, at different stages of development, in response to different environmental or physiological conditions, and/or in different species. In one aspect, the promoter functions as a switch to activate the expression of a gene.
In one aspect, the promoter is “constitutive.” A constitutive promoter is a promoter that causes a gene to be expressed in most cell types at most times. In another aspect, the promoter is “regulated.” A regulated promoter is a promoter that becomes active in response to a specific stimulus. A promoter may be regulated chemically, such as, for example, in response to the presence or absence of a particular metabolite (e.g., lactose or tryptophan), a metal ion, a molecule secreted by a pathogen, or the like. A promoter also may be regulated physically, such as, for example, in response to heat, cold, water stress, salt stress, oxygen concentration, illumination, wounding, or the like.
Promoters that are useful to drive expression of the nucleotide sequences described herein are numerous and familiar to those skilled in the art. Suitable promoters include, but are not limited to, the following: T3 promoter, T7 promoter, an iron promoter, and GAL1 promoter. In a further aspect, the promoter is a native part of the vector used herein. Variants of these promoters are also contemplated. The skilled artisan will be able to use site-directed mutagenesis and/or other mutagenesis techniques to modify the promoters to promote more efficient function. The promoter may be positioned, for example, from 10-100 nucleotides from a ribosomal binding site.
In one aspect, the promoter is a GAL1 promoter. In another aspect, the GAL1 promoter is native to the plasmid used to create the vector. In another aspect, a GAL1 promoter is positioned before the a gene that expresses hexokinase, the gene that expresses a heat shock protein, the gene that expresses geranylgeranyl pyrophosphate synthase 2, the gene that expresses 1-deoxy-
In another aspect, the regulatory sequence is a terminator or stop sequence. As used herein, a terminator is a sequence of DNA that marks the end of a gene or operon to be transcribed. In a further aspect, the terminator is an intrinsic terminator or a Rho-dependent transcription terminator. As used herein, an intrinsic terminator is a sequence wherein a hairpin structure can form in the nascent transcript that disrupts the mRNA/DNA/RNA polymerase complex. As used herein, a Rho-dependent transcription terminator requires a Rho factor protein complex to disrupt the mRNA/DNA/RNA polymerase complex. In one aspect, the terminator is a T7 terminator. In an alternative aspect, the terminator is a CYC1 terminator obtained from or native to the pYES2 plasmid.
In a further aspect, the regulatory sequence includes both a promoter and a terminator or stop sequence. In a still further aspect, the regulatory sequence can include multiple promoters or terminators. Other regulatory elements, such as enhancers, are also contemplated. Enhancers may be located from about 1 to about 2000 nucleotides in the 5′ direction from the start codon of the DNA to be transcribed, or may be located 3′ to the DNA to be transcribed. Enhancers may be “cis-acting,” that is, located on the same molecule of DNA as the gene whose expression they affect.
In one aspect, when the vector is a plasmid, the plasmid can also contain a multiple cloning site or polylinker. In a further aspect, the polylinker contains recognition sites for multiple restriction enzymes. The polylinker can contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 recognition sites for restriction enzymes. Further, restriction sites may be added, disabled, or removed as required, using techniques known in the art. In one aspect, the plasmid contains restriction sites for any known restriction enzyme such as, for example, HindIII, KpnI, SacI, BamHI, BstXI, EcoRI, BasBI, NotI, XhoI, SphI, XbaI, ApaI, SalI, ClaI, EcoRV, PstI, SmaI, XmaI, SpeI, EagI, SacII, or any combination thereof. In a further aspect, the plasmid contains more than one recognition site for the same restriction enzyme.
In one aspect, the restriction enzyme can cleave DNA at a palindromic or an asymmetrical restriction site. In a further aspect, the restriction enzyme cleaves DNA to leave blunt ends; in an alternative aspect, the restriction enzyme cleaves DNA to leave “sticky” or overhanging ends. In another aspect, the enzyme can cleave DNA to a distance of from 20 bases to over 1000 bases away from the restriction site. A variety of restriction enzymes are commercially available and their recognition sequences, as well as instructions for use (e.g., amount of DNA needed, precise volumes or reagents, purification techniques, as well as information about salt concentration, pH, optimum temperature, incubation time, and the like) are provided by enzyme manufacturers.
In one aspect, a plasmid with a polylinker containing one or more restriction sites can be digested with one restriction enzyme and a nucleotide sequence of interest can be ligated into the plasmid using a commercially-available DNA ligase enzyme. Several such enzymes are available, often as kits containing all reagents and instructions required for use. In another aspect, a plasmid with a polylinker containing two or more restriction sites can be simultaneously digested with two restriction enzymes and a nucleotide sequence of interest can be ligated into the plasmid using a DNA ligase enzyme. Using two restriction enzymes provides an asymmetric cut in the DNA, allowing for insertion of a nucleotide sequence of interest in a particular direction and/or on a particular strand of the double-stranded plasmid. Since RNA synthesis from a DNA template proceeds from 5′ to 3′, usually starting just after a promoter, the order and direction of elements inserted into a plasmid can be especially important. If a plasmid is to be simultaneously digested with multiple restriction enzymes, these enzymes must be compatible in terms of buffer, salt concentration, and other incubation parameters.
In some aspects, prior to ligation using a ligase enzyme, a plasmid that has been digested with a restriction enzyme is treated with an alkaline phosphatase enzyme to remove 5′ terminal phosphate groups. This prevents self-ligation of the plasmid and thus facilitates ligation of heterologous nucleotide fragments into the plasmid.
In one aspect, different genes can be ligated into a plasmid in one pot. In this aspect, the genes will first be digested with restriction enzymes. In certain aspects, the digestion of genes with restriction enzymes provides multiple pairs of matching 5′ and 3′ overhangs that will spontaneously assemble the genes in the desired order. In another aspect, the genes and components to be incorporated into a plasmid can be assembled into a single insert sequence prior to insertion into the plasmid. In a further aspect, a DNA ligase enzyme can be used to assist in the ligation process.
In another aspect, the ligation mix may be incubated in an electromagnetic chamber. In one aspect, this incubation lasts for about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 1 hour.
The DNA construct described herein can be part of a vector. In a further aspect, the vector is a plasmid, a phagemid, a cosmid, a yeast artificial chromosome, a bacterial artificial chromosome, a virus, a phage, or a transposon. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with the hosts. The vector ordinarily carries a replication site as well as marking sequences that are capable of performing phenotypic selection in transformed cells. Plasmid vectors are well known and are commercially available. Such vectors include, but are not limited to, pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pBR322, pYES, pYES2, pBSKII, pUC, and pUC19 vectors.
Plasmids are double-stranded, autonomously-replicating, genetic elements that are not integrated into host cell chromosomes. Further, these genetic elements are usually not part of the host cell's central metabolism. In bacteria, plasmids may range from 1 kilobase (kb) to over 200 kb. Plasmids can be engineered to encode a number of useful traits including the production of secondary metabolites, antibiotic resistance, the production of useful proteins, degradation of complex molecules and/or environmental toxins, and others. Plasmids have been the subject of much research in the field of genetic engineering, as plasmids are convenient expression vectors for foreign DNA in, for example, microorganisms. Plasmids generally contain regulatory elements such as promoters and terminators and also usually have independent replication origins. Ideally, plasmids will be present in multiple copies per host cell and will contain selectable markers (such as genes for antibiotic resistance) to allow the skilled artisan to select host cells that have been successfully transfected with the plasmids (for example, by growing the host cells in a medium containing the antibiotic).
Vectors capable of high levels of expression of recombinant genes and proteins are well known in the art. Vectors useful for the transformation of a variety of host cells are common and commercially available and include. The skilled practitioner will be able to choose a plasmid based on such factors as a) the amount of nucleic acid (i.e., number of genes and other elements) to be inserted, b) the host organism, c) culture conditions for the host organism, and other related factors.
In one aspect, the vector encodes a selection marker. In a further aspect, the selection marker is a gene that confers resistance to an antibiotic. In certain aspects, during fermentation of host cells transformed with the vector, the cells are contacted with the antibiotic. For example, the antibiotic may be included in the culture medium. Cells that have not been successfully transformed cannot survive in the presence of the antibiotic; only cells containing the vector which confers antibiotic resistance can survive. Optimally, only cells containing the vector to be expressed will be cultured, as this will result in the highest production efficiency of the desired gene products (e.g., peptides). Cells that do not contain the vector would otherwise compete with transformed cells for resources. In one aspect, the antibiotic is tetracycline, neomycin, kanamycin, ampicillin, hygromycin, chloramphenicol, amphotericin B, bacitracin, carbapenam, cephalosporin, ethambutol, fluoroquinolones, isonizid, methicillin, oxacillin, vancomycin, streptomycin, quinolines, rifampin, rifampicin, sulfonamides, cephalothin, erythromycin, streptomycin, gentamycin, penicillin, other commonly-used antibiotics, or a combination thereof.
In certain aspects, the DNA construct can include a gene that expresses a reporter protein. The selection of the reporter protein can vary. For example, the reporter protein can be a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In one aspect, the reporter protein is a yellow fluorescent protein and the gene that expresses the reporter protein has SEQ ID NO. 9 or at least 70% homology thereto, at least 75% homology thereto, at least 80% homology thereto, at least 85% homology thereto, at least 90% homology thereto, or at least 95% homology thereto. The amount of fluorescence that is produced by the biological device can be correlated to the amount of DNA incorporated into the plant cells. The fluorescence produced by the device can be detected and quantified using techniques known in the art. For example, spectrofluorometers are typically used to measure fluorescence. The Examples provide exemplary procedures for measuring the amount of fluorescence as a result of the expression of DNA.
(c) a gene that expresses mevalonate pyrophosphate decarboxylase; (d) a gene that expresses hexokinase; (e) a gene that expresses HSP70; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2; and (g) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses 4-hydroxy-3-methylbut-2-enyl diphosphate reductase having SEQ ID NO. 1 or at least 70% homology thereto; (b) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (c) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (d) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; and (e) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a GAL1 promoter; (b) a gene that expresses 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; (c) a CYC1 terminator; (d) a GAL1 promoter; (e) a gene that expresses hexokinase; (f) a CYC1 terminator; (g) a GAL1 promoter; (h) a gene that expresses HSP70; (i) a CYC1 terminator; (j) a GAL1 promoter; (k) a gene that expresses geranylgeranyl pyrophosphate synthase 2; (l) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses 1-deoxy-
In still another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a GAL1 promoter; (b) a gene that expresses 4-hydroxy-3-methylbut-2-enyl diphosphate reductase having SEQ ID NO. 1 or at least 70% homology thereto; (c) a CYC1 terminator; (d) a GAL1 promoter; (e) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (f) a CYC1 terminator; (g) a GAL1 promoter; (h) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (i) a CYC1 terminator; (j) a GAL1 promoter; (k) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (1) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase; (b) a gene that expresses isopentenyl pyrophosphate isomerase; (c) a gene that expresses mevalonate pyrophosphate decarboxylase; (d) a gene that expresses hexokinase; (e) a gene that expresses HSP70; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2; and (g) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (c) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (d) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (e) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; and (g) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a GAL1 promoter; (b) a gene that expresses mevalonate-5-kinase; (c) a CYC1 terminator; (d) a GAL1 promoter; (e) a gene that expresses isopentenyl pyrophosphate isomerase; (f) a CYC1 terminator; (g) a GAL1 promoter, (h) a gene that expresses mevalonate pyrophosphate decarboxylase; (i) a CYC1 terminator; (j) a GAL1 promoter; (k) a gene that expresses hexokinase; (1) a CYC1 terminator; (m) a GAL1 promoter; (n) a gene that expresses HSP70; (o) a CYC1 terminator; (p) a GAL1 promoter; (q) a gene that expresses geranylgeranyl pyrophosphate synthase 2; (r) a CYC1 terminator; (s) a GAL1 promoter; (t) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a GAL1 promoter; (b) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (c) a CYC1 terminator; (d) a GAL1 promoter; (e) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (f) a CYC1 terminator; (g) a GAL1 promoter, (h) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (i) a CYC1 terminator; (j) a GAL1 promoter; (k) a gene that expresses having hexokinase SEQ ID NO. 2 or at least 70% homology thereto; (1) a CYC1 terminator; (m) a GAL1 promoter; (n) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (o) a CYC1 terminator; (p) a GAL1 promoter; (q) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (r) a CYC1 terminator; (s) a GAL1 promoter; (t) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (c) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (d) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (e) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (g) a gene that expresses a UDP-glycosyltransferase having SEQ ID NO. 12 or at least 70% homology thereto; and (h) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses HSP70; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses UGT76G1His155Leu, a CYC1 terminator, (u) a GAL1 promoter, and (v) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses UGT76G1His155Leu having SEQ ID NO. 12 or at least 70% homology thereto; (t) a CYC1 terminator, (u) a GAL1 promoter, and (v) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (c) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (d) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (e) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (g) a gene that expresses an O-linked acetylglucosamine transferase having SEQ ID NO. 13 or at least 70% homology thereto; and (h) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses HSP70; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses an O-linked acetylglucosamine transferase; (t) a CYC1 terminator; (u) a GAL1 promoter; and (v) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses HSP70 having SEQ ID NO. 3 or at least 70% homology thereto; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses an O-linked acetylglucosamine transferase having SEQ ID NO. 13 or at least 70% homology thereto; (t) a CYC1 terminator; (u) a GAL1 promoter; and (v) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (c) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (d) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (e) a gene that expresses an O-linked acetylglucosamine transferase having SEQ ID NO. 13 or at least 70% homology thereto; (f) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (g) a gene that expresses UGT76G1His155Leu having SEQ ID NO. 12 or at least 70% homology thereto; and (h) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses an O-linked acetylglucosamine transferase; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses UGT76G1His155Leu a CYC1 (t) terminator; (u) a GAL1 promoter; and (v) a gene that expresses 1-deoxy-
In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses mevalonate-5-kinase having SEQ ID NO. 6 or at least 70% homology thereto; (b) a CYC1 terminator; (c) a GAL1 promoter; (d) a gene that expresses isopentenyl pyrophosphate isomerase having SEQ ID NO. 7 or at least 70% homology thereto; (e) a CYC1 terminator; (f) a GAL1 promoter, (g) a gene that expresses mevalonate pyrophosphate decarboxylase having SEQ ID NO. 8 or at least 70% homology thereto; (h) a CYC1 terminator; (i) a GAL1 promoter; (j) a gene that expresses hexokinase having SEQ ID NO. 2 or at least 70% homology thereto; (k) a CYC1 terminator; (1) a GAL1 promoter; (m) a gene that expresses an O-linked acetylglucosamine transferase having SEQ ID NO. 13 or at least 70% homology thereto; (n) a CYC1 terminator; (o) a GAL1 promoter; (p) a gene that expresses geranylgeranyl pyrophosphate synthase 2 having SEQ ID NO. 4 or at least 70% homology thereto; (q) a CYC1 terminator; (r) a GAL1 promoter; (s) a gene that expresses UGT76G1His155Leu having SEQ ID NO. 12 or at least 70% homology thereto; (t) a CYC1 terminator; (u) a GAL1 promoter; and (v) a gene that expresses 1-deoxy-
In one aspect, the DNA construct has SEQ ID NO. 10 (
In one aspect, a “biological device” is formed when a microbial cell is transfected with the DNA construct described herein. The biological devices are generally composed of microbial host cells, where the host cells are transformed with a DNA construct described herein.
In one aspect, the DNA construct is carried by the expression vector into the cell and is separate from the host cell's genome. In another aspect, the DNA construct is incorporated into the host cell's genome. In still another aspect, incorporation of the DNA construct into the host cell enables the host cell to produce steviol glycosides. “Heterologous” genes and proteins are genes and proteins that have been experimentally inserted into a cell that are not normally expressed by the cell. A heterologous gene may be cloned or derived from a different cell type or species than the recipient cell or organism. Heterologous genes may be introduced into cells by transduction or transformation.
An “isolated” nucleic acid is one that has been separated from other nucleic acid molecules and/or cellular material (peptides, proteins, lipids, saccharides, and the like) normally present in the natural source of the nucleic acid. An “isolated” nucleic acid may optionally be free of the flanking sequences found on either side of the nucleic acid as it naturally occurs. An isolated nucleic acid can be naturally occurring, can be chemically synthesized, or can be a cDNA molecule (i.e., is synthesized from an mRNA template using reverse transcriptase and DNA polymerase enzymes).
“Transformation” or “transfection” as used herein refers to a process for introducing heterologous DNA into a host cell. Transformation can occur under natural conditions or may be induced using various methods known in the art. Many methods for transformation are known in the art and the skilled practitioner will know how to choose the best transformation method based on the type of cells being transformed. Methods for transformation include, for example, viral infection, electroporation, lipofection, chemical transformation, and particle bombardment. Cells may be stably transformed (i.e., the heterologous DNA is capable of replicating as an autonomous plasmid or as part of the host chromosome) or may be transiently transformed (i.e., the heterologous DNA is expressed only for a limited period of time).
“Competent cells” refers to microbial cells capable of taking up heterologous DNA. Competent cells can be purchased from a commercial source, or cells can be made competent using procedures known in the art. Exemplary procedures for producing competent cells are provided in the Examples.
The host cells as referred to herein include their progeny, which are any and all subsequent generations formed by cell division. It is understood that not all progeny may be identical due to deliberate or inadvertent mutations. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
A transformed cell includes the primary subject cell and its progeny. The host cells can be naturally-occurring cells or “recombinant” cells. Recombinant cells are distinguishable from naturally-occurring cells in that naturally-occurring cells do not contain heterologous DNA introduced through molecular cloning procedures. In one aspect, the host cell is a prokaryotic cell such as, for example, Escherichia coli. In other aspects, the host cell is a eukaryotic cell such as, for example, the yeast Saccharomyces cerevisiae. Host cells transformed with the DNA construct described herein are referred to as “biological devices.”
The DNA construct is first delivered into the host cell. In one aspect, the host cells are naturally competent (i.e., able to take up exogenous DNA from the surrounding environment). In another aspect, cells must be treated to induce artificial competence. This delivery may be accomplished in vitro, using well-developed laboratory procedures for transforming cell lines. Transformation of bacterial cell lines can be achieved using a variety of techniques. One method involves calcium chloride. The exposure to the calcium ions renders the cells able to take up the DNA construct. Another method is electroporation. In this technique, a high-voltage electric field is applied briefly to cells, producing transient holes in the membranes of the cells through which the vector containing the DNA construct enters. Another method involves exposing intact yeast cells to alkali cations such as, for example, lithium. In one aspect, this method includes exposing yeast to lithium acetate, polyethylene glycol, and single-stranded DNA such as, for example, salmon sperm DNA. Without wishing to be bound by theory, the single-stranded DNA is thought to bind to the cell wall of the yeast, thereby blocking plasmids from binding. The plasmids are then free to enter the yeast cell. Enzymatic and/or electromagnetic techniques can also be used alone, or in combination with other methods, to transform microbial cells. Exemplary procedures for transforming yeast and bacteria with specific DNA constructs are provided in the Examples. In certain aspects, two or more types of DNA can be incorporated into the host cells. Thus, different metabolites can be produced from the same host cells at enhanced rates.
The biological devices described herein are useful in the production of steviol glycosides. Once the DNA construct has been incorporated into the host cell, the cells are cultured such that the cells multiply. A satisfactory microbiological culture contains available sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts, and, in certain cases, vitamins or other growth-promoting substances. For example, the addition of peptone provides a readily-available source of nitrogen and carbon. Furthermore, the use of different types of media results in different growth rates and different stationary phase densities; stationary phase is where secondary metabolite production occurs most frequently. A rich media results in a short doubling time and higher cell density at stationary phase. Minimal media results in slow growth and low final cell densities. Efficient agitation and aeration increase final cell densities.
In one aspect, host cells can be cultured or fermented by any method known in the art. The skilled practitioner will be able to select a culture medium based on the species and/or strain of host cell selected. In certain aspects, the culture medium will contain a carbon source. A variety of carbon sources are contemplated, including, but not limited to: monosaccharides such as glucose and fructose, disaccharides such as lactose or sucrose, oligosaccharides, polysaccharides such as starch, or mixtures thereof. In one aspect, the biological devices described herein are cultured with a medium composed of raffionose, galactose, or a combination thereof. Unpurified mixtures extracted from feedstocks are also contemplated and include molasses, barley malt, and related compounds and compositions. Other glycolytic and tricarboxylic acid cycle intermediates are also contemplated as carbon sources, as are one-carbon substrates such as carbon dioxide and/or methanol in the cases of compatible organisms. The carbon source utilized is limited only by the particular organism being cultured.
Culturing or fermenting of host cells can be accomplished by any technique known in the art. In one aspect, batch fermentation can be conducted. In batch fermentation, the composition of the culture medium is set at the beginning and the system is closed to future alterations. In some aspects, a limited form of batch fermentation may be carried out, wherein factors such as oxygen concentration and pH are manipulated, but additional carbon is not added. Continuous fermentation methods are also contemplated. In continuous fermentation, equal amounts of a defined medium are continuously added to and removed from a bioreactor. In other aspects, microbial host cells are immobilized on a substrate. Fermentation may be carried out on any scale and may include methods in which literal “fermentation” is carried out as well as other culture methods that are non-fermentative.
In one aspect, the method involves growing the biological devices described herein for a sufficient time to produce steviol glycosides. The ordinary artisan will be able to choose a culture medium and optimum culture conditions based on the biological identity of the host cells.
In certain aspects, after culturing the biological device to produce the steviol glycoside, the host cells of the device can be lysed with one or more enzymes. For example, when the host cells are yeast, the yeast cells can be lysed with lyticase. In one aspect, the lyticase concentration can be 500, 600, 700, 800, 900, or 1000 μL per liter of culture, where any value can be the lower and upper end-point of a range (e.g., 500 to 900 μL, 600 to 800 μL, etc.).
In addition to enzymes, other components can be used to facilitate lysis of the host cells. In one aspect, chitosan can be used in combination with an enzyme to lyse the host cells. Chitosan is generally composed of glucosamine units and N-acetylglucosamine units and can be chemically or enzymatically extracted from chitin, which is a component of arthropod exoskeletons and fungal and microbial cell walls. In certain aspects, the chitosan can be acetylated to a specific degree of acetylation in order to enhance tissue growth during culturing as well as metabolite production. In one aspect, the chitosan is from 60% to about 100%, 70% to 90%, 75% to 80%, or about 80% acetylated. The molecular weight of the chitosan can vary, as well. For example, the chitosan can comprise about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glucosamine units and/or N-acetylglucosamine units. In another aspect, the chitosan includes 5 to 7 glucosamine units and/or N-acetylglucosamine units. In one aspect, chitosan can be added until a concentration of 0.0015, 0.0025, 0.005, 0.0075, 0.01, 0.015, 0.02, 0.03, 0.04, or 0.05 (where any value can be a lower and upper end-point of a range, e.g., 0.005 to 0.02, 0.0075 to 0.015, etc.) is achieved in the culture. Still further in this aspect, the chitosan is present at a concentration of 0.01%.
In a further aspect, the steviol glycoside can be collected, separated from the microbial cells (lysed or intact), and/or purified through any technique known in the art such as, for example, precipitation, centrifugation, filtration, and the like. In one aspect, the steviol glycoside can be purified via microfiltration to remove impurities. In one aspect, the microfilter has a pore size of 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, or 0.8 μm, where any value can be a lower and/or upper end-point of a range (e.g., 0.3 μm to 0.5 μm).
In another aspect, the steviol glycoside can be chemically-modified to produce additional desirable properties. Alternatively, compositions composed of the steviol glycosides with lysed and/or intact host cells (e.g., yeast) can be used herein, where it is not necessary to separate the host cells and other components from the steviol glycosides.
The steviol glycoside produced from the devices herein can be a mixture of two or more compounds. Examples of steviol glycosides produced herein include, but are not limited to, stevioside, steviol, rubusoside, steviol-13-O-glucoside, steviol-19-O-glucoside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, and dulcoside A. Individual compounds can be isolated and purified or, in the alternative, mixtures of tow or more compounds can be used.
The biological devices described herein in addition to producing steviol glycosides produce other sugars. In one aspect, the biological devices produced increased amounts of sucrose. Certain steviol glycosides possess a bitter taste. The biological devices described herein produce sucrose in a sufficient amount to mask the bitter taste of the steviol glycosides.
In one aspect, provided herein is a method for producing one or more steviol glycosides from plant cells, where the method involves contacting the plant cells with the biological device disclosed herein.
The selection of the plant used in the methods described herein can vary depending on the application. For example, a specific plant can be selected that produces certain desirable metabolites. An example of one such metabolite is rebaudioside A, or rebA. RebA is 200 times sweeter than sugar and has numerous applications in the food industry (e.g., natural non-caloric sweeteners), pharmaceuticals (e.g., improving the flavor of medications), and dietary supplements. Current techniques for producing steviol glycosides are expensive. For example, large amounts of fresh plant biomass must be cultivated and harvested and expensive and time-consuming extraction methods must be used. The biological devices and methods described herein enhance the production of steviol glycosides from plants that naturally produce steviol glycosides. In one aspect, the plant can include, but is not limited to, Stevia rebaudiana, Stevia phlebophylla, or Rubus chingii.
In another aspect, other steviol glycosides in addition to rebA can have the same or similar food, pharmaceutical, and dietary supplement applications described above with respect to rebA.
In one aspect, plant cells when contacted with the biological devices described above exhibit enhanced production of rebA and/or other steviol glycosides. Recipient cell targets include, but are not limited to, meristem cells, Type I, Type II, and Type III callus, immature embryos and gametic cells such as microspores, pollen, sperm, and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Type I, Type II, and Type III callus may be initiated from tissue sources including, but not limited to, immature embryos, immature inflorescences, seedling apical meristems, microspores, and the like. Those cells that are capable of proliferating as callus are also useful herein. Methods for growing plant cells are known in the art (see U.S. Pat. No. 7,919,679). Exemplary procedures for growing plant calluses are also provided in the Examples. In one aspect, plant calluses grown from 2 to 4 weeks can be used herein. The plant cells can also be derived from plants varying in age. For example, plants that are 80 days to 120 days old after pollination can be used to produce calluses useful herein.
The plant cells can be contacted with the biological device in a number of different ways. In one aspect, the device can be added to media containing the plant cells. In another aspect, the device can be injected into the plant cells via syringe. The amount of device and the duration of exposure to the device can vary as well. In one aspect, the concentration of the device is about 103, 104, 105, 106, 107, 108, or 109 cells/mL of water. In one aspect, when the host cell is a bacterium, the concentration of the device is 106. In another aspect, when the host cell is yeast, the concentration of the device is 109. Different volumes of the biological device can be used as well, ranging from 5 μL to 500 μL.
Once the plant cells have been in contact with the biological device for a sufficient time to produce the metabolite (e.g., rebA or another steviol glycoside), the metabolite is isolated. In one aspect, the metabolite is extracted from the media containing the biological device and the plant cells. The selection of the extraction solvent can vary depending upon the solubility of the metabolite.
With current techniques, the extraction of metabolites produced from plants usually requires high initial amounts of plant biomass or material, which in turn requires larger amounts of extraction solvents. The use of higher amounts of extraction solvents adds to the expense of metabolite production. The use of higher amounts of organic solvents presents environmental risks as well. However, the use of the biological devices described herein produces significantly higher amounts of metabolites such as rebA and other steviol glycosides, which means smaller amounts of biomass are required in order to produce and isolate the metabolites when compared with existing techniques. The extraction of plant metabolites using current techniques also requires fresh biomass, which entails agronomic practices, the use of chemicals, and time-consuming extraction methods. Therefore, the use of the biological devices described herein is more cost-effective and safer for the environment than traditional methods for producing and synthesizing steviol glycosides.
In certain aspects, any of the biological devices described above can be used in combination with a polysaccharide. In one aspect, the plant cells are first contacted with the biological device then subsequently contacted with the polysaccharide. In another aspect, the plant cells are first contacted with the polysaccharide then subsequently contacted with the biological device. In a further aspect, the plant cells are only contacted with a polysaccharide and not contacted with the biological device. In a still further aspect, the plant cells are contacted simultaneously with the polysaccharide and the biological device.
In one aspect, the polysaccharide includes chitosan, glucosamine (GlcN), N-acetylglucosamine (NAG), or any combination thereof. Chitosan is generally composed of GlcN and NAG units and can be chemically or enzymatically extracted from chitin, which is a component of arthropod exoskeletons and fungal and microbial cell walls. In certain aspects, the chitosan can be acetylated to a specific degree of acetylation in order to enhance tissue growth during culturing as well as metabolite production. In one aspect, the chitosan is from about 60% to about 100%, 70% to 90%, 75% to 85%, or about 80% acetylated. Exemplary procedures for producing and isolating the chitosan are provided in the Examples. In one aspect, chitosan isolated from shells of crab, shrimp, lobster, and/or krill is useful herein.
The molecular weight of the chitosan can vary, as well. For example, the chitosan comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GlcN and/or NAG units. In another aspect, the chitosan includes 5 to 7 GlcN and/or NAG units. In one aspect, the chitosan is in a solution of water and acetic acid at less than 1% by weight, less than 0.75% by weight, less than 0.5% by weight, less than 0.25% by weight, or less than 0.1% by weight. In another aspect, the amount of chitosan that is applied to the plant cells is from 0.1% to 0.01% by weight, from 0.075% to 0.025% by weight, or is about 0.05% by weight. The polysaccharides used herein are generally natural polymers and thus present no environmental concerns. Additionally, the polysaccharide can be used in acceptably low concentrations. In certain aspects, the polysaccharide can be used in combination with one or more growth regulators.
In one aspect, the plant growth regulator is an auxin, a cytokinin, a gibberellin, abscisic acid, or a polyamine. In a further aspect, the auxin is a natural or synthetic auxin. In a still further aspect, the auxin is indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), 2-phenylacetic acid (PAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (α-NAA), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (torden or picloram), 2,4,5-trichloropicolinic acid (2,4,5-T), or a combination thereof. In another aspect, the cytokinin is zeatin, kinetin, 6-benzylaminopurine, diphenylurea, thidizuron (TDZ), 6-(γ,γ-dimethylallylamino)purine, or a combination thereof. In another aspect, the gibberellins is gibberellins A1 (GA1), gibberellic acid (GA3), ent-gibberellane, ent-kaurene, or a combination thereof. In yet another aspect, the polyamine is putrescine, spermidine, or a combination thereof.
In one aspect, the plant cell or callus is first contacted with a polysaccharide and subsequently contacted with a plant growth regulator. In another aspect, the plant cell or callus is first contacted with a plant growth regulator and subsequently contacted with a polysaccharide. In an alternative aspect, the plant cell or callus is simultaneously contacted with a polysaccharide and a plant growth regulator. In a further aspect, the plant cell or callus is only contacted with a polysaccharide and is not contacted with a plant growth regulator.
The plant cells can be contacted with the polysaccharide using a number of techniques. In one aspect, the plant cells or reproductive organs (e.g., a plant embryo) can be cultured in agar and medium with a solution of the polysaccharide. In other aspects, the polysaccharide can be applied to a plant callus by techniques such as, for example, coating the callus or injecting the polysaccharide into the callus. In this aspect, the age of the callus can vary depending on the type of plant. The amount of polysaccharide can vary depending upon, among other things, the selection and number of plant cells. The use of the polysaccharide in the methods described herein permits rapid tissue culturing at room temperature. Due to the ability of the polysaccharide to prevent microbial contamination, the tissue culture can grow for extended periods of time ranging from days to several weeks. Moreover, tissue culturing with the polysaccharide can occur in the dark and/or light. As discussed above, the plant cells can optionally be contacted with any of the biological devices described above. Thus, the use of the polysaccharides and biological devices described herein is a versatile way to culture and grow plant cells—and, ultimately, plants of interest—with enhanced physiological properties.
In other aspects, the plant cells can be cultured in a liquid medium on a larger scale in a bioreactor. For example, plant cells can be cultured in agar and medium, then subsequently contacted with (e.g., injected) a biological device described herein. After a sufficient culturing time (e.g., two to four weeks), the plant cells are introduced into a container with the same medium used above and, additionally, the polysaccharide. In certain aspects, the polysaccharide can be introduced with anionic polysaccharides including, but not limited to, alginates (e.g., sodium, calcium, potassium, etc.). After the introduction of the polysaccharide, the solution is mixed for a sufficient time to produce a desired result (e.g., production of a desired metabolite).
In one aspect, provided herein is a plant grown by the process consisting of contacting plant gamete cells or a plant reproductive organ with the biological devices disclosed herein. In a further aspect, the plant is produced by the following method:
In one aspect, the plant is Stevia rebaudiana, Stevia phlebophylla, or Rubus chingii. In a further aspect, the method of growing the plant described above includes an additional step (d), wherein the plant callus is cultured with chitosan.
In one aspect, provided herein is a method for producing one or more steviol glycosides from plant cells, the method including the steps of:
In an alternative aspect, provided herein is a method for producing one or more steviol glycosides from leaves, the method including the steps of:
In a further aspect, the same method can be applied to other plant parts including fruits, stems, roots, tubers, corms, bulbs, flowers, buds, seeds, and the like. In a still further aspect, the same method can be applied to an entire plant.
In one aspect, the plant callus is immersed in a solution of polysaccharide (e.g., chitosan) then inoculated with the device. In one aspect, the plant callus is that of Stevia rebaudiana, Stevia phlebophylla, or Rubus chingii. The plant callus can be from 2 days up to 20 days old prior to inoculation with the device. The plant callus is then allowed to grow until it is of sufficient weight and size. In one aspect, the plant callus is allowed to grow (i.e., culture) for 1 to 10 weeks after inoculation. The next step involves removal of the rebA or other steviol glycoside from the callus.
In another aspect, a plant callus described above can be planted and allowed to grow and mature into a plant bearing fruit and leaves. In one aspect, rebA or another steviol glycoside can be isolated from a plant that has been grown from a plant callus inoculated with a device described herein and optionally contacted with a polysaccharide (e.g., chitosan). In one aspect, the steviol glycoside can be removed from the leaves of a plant grown with the devices described herein. In one aspect, the leaves of Stevia rebaudiana grown from calluses inoculated with the devices described herein provide a rich source of rebA.
The ordinarily-skilled artisan will be able to use established procedures for culturing and providing nutrients to the calluses. In one aspect, calluses ranging in age from one to four weeks, or about 7, 14, 15, 20, 21, 25, or 28 days can be used in the procedures described herein. Further in this aspect, calluses can be inoculated with biological devices and, optionally, chitosan at the start of steviol glycoside production. In one aspect, 15-day-old calluses (e.g., 15 days post-inoculation) are used. In a further aspect, the calluses can be placed under an artificial light source in a chamber where conditions such as temperature and humidity are controlled. At various points during callus culture, calluses can be transferred to trays with fresh nutrients or can be directly sprayed with fresh nutrients.
In one aspect, calluses can be grown in trays for a period of time ranging from 1 to 6 months, or can be grown for 1, 2, 3, 4, 5, or 6 months. In one aspect, the calluses are grown for 3 months. Further in this aspect, the calluses have generally sprouted small plants after 3 months of growth.
Several procedures have been established for extraction of hydrophobic compounds such as steviol glycosides from calluses, or the following procedures can be used. In one aspect, callus samples are lyophilized and weighed. Further in this aspect, the samples are placed in ethyl acetate at a ratio of from 1:50 to 1:200 of callus:ethyl acetate (w:v) and macerated. In one aspect, the ratio is 1:100. In a further aspect, callus samples that have been homogenized can be sealed and placed in a water bath with shaking.
Alternatively, steviol glycosides can be extracted from leaves. In a further aspect, the procedure for extracting steviol glycosides from leaves is the same for extracting from calluses, with the exception of the leaf:solvent ratio, which can be from 1:1 to 1:20. In one aspect, the leaf:ethyl acetate ratio is 1:10.
In a further aspect, any solvent in which rebA or the desired steviol glycoside is soluble in can be used in place of a portion or all of the ethyl acetate in the procedure described above. In a further aspect, the solvent can be hexane, methanol, acetone, dichloromethane, chloroform, ethanol, diethyl ether, DMSO, toluene, isopropyl alcohol, n-butane, heptanes, acetonitrile, THF, or a combination thereof.
In one aspect, steviol glycosides such as, for example, rebA, stevioside, and other steviol glycosides produced by the devices and methods described above can be quantified and/or purified using high pressure liquid chromatography (HPLC). Exemplary methods for quantifying steviol glycosides are provided in the examples.
The extracts composed of steviol glycosides produced herein can be used to make food products, dietary supplements and sweetener compositions. For example, the steviol glycoside can be included in food products such as ice cream, beverages, (e.g., carbonated fruit juices, energy drinks), yogurts, baked goods, chewing gums, hard and soft candies, sauces, and tabletop sweeteners. The extracts can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements. The extracts can also be included in animal feed products for both the agriculture industry and the companion animal industry.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. Numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and conditions can be used to optimize the produce purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such processes and conditions.
The DNA construct was composed of genetic components described herein and assembled in plasmid vectors (e.g., pYES2 and pBSK). Sequences of genes and/or proteins with desired properties were identified in GenBank; these included a DXP synthase gene, a beta-carotene hydroxylase gene, and a lycopene epsilon-cyclase gene. Other genetic parts were also obtained for inclusion in the DNA constructs including, for example, promoter genes (e.g., GAL1 promoter), reporter genes (e.g., yellow fluorescent reporter protein), and terminator sequences (e.g., CYC1 terminator). These genetic parts included restriction sites for ease of insertion into plasmid vectors.
The cloning of the DNA construct into the biological devices was performed as follows. Sequences of individual genes were amplified by polymerase chain reaction using primers that incorporated restriction sites at their 5′ ends to facilitate construction of the full sequence to be inserted into the plasmid. Genes were then ligated using standard protocols to form an insert. The plasmid was then digested with restriction enzymes according to directions and using reagents provided by the enzyme's supplier (Promega). The complete insert, containing restriction sites on each end, was then ligated into the plasmid. Successful construction of the insert and ligation of the insert into the plasmid were confirmed by gel electrophoresis.
PCR was used to enhance DNA concentration using a Mastercycler Personal 5332 ThermoCycler (Eppendorf North America) with specific sequence primers and the standard method for amplification (Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.). Digestion and ligation were used to ensure assembly of DNA synthesized parts using restriction enzymes and reagents (PCR master mix of restriction enzymes: XhoI, KpnI, XbaI, EcoRI, BamHI, and HindIII, with alkaline phosphatase and quick ligation kit, all from Promega). DNA was quantified using a NanoVue spectrophotometer (GE Life Sciences) and a standard UV/Visible spectrophotometer using the ratio of absorbances at 260 nm versus 280 nm. In order to verify final ligations, DNA was visualized and purified via electrophoresis using a Thermo EC-150 power supply.
The DNA construct was made with gene parts fundamental for expression of sequences such as, for example, ribosomal binding sites, native and constitutive promoters, reporter genes, and transcriptional terminators or stops. Backbone plasmids and synthetic inserts can be mixed together for ligation purposes at different ratios ranging from 1:1, 1:2, 1:3, 1:4, and up to 1:5. In one aspect, the ratio of backbone plasmid to synthetic insert is 1:4. The DNA constructs in
After the vector comprising the DNA construct has been produced, the resulting vector can be incorporated into the host cells using the methods known in the art (e.g., Gietz, R. D. and R. H. Schiestl, 2007, Nature Protocols, “Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method,” Vol. 2, 35-37, doi:10.1038/nprot.2007.14).
The steviol glycosides rebaudioside A (rebA) and stevioside produced by yeast transformed independently with SEQ ID NOs. 15 and 16 using the procedure in Example 1 were quantified using HPLC. Chromatographic separations were carried out on a Thermo Scientific Dionex Ultimate 3000 UHPLC system, using a Thermo Scientific variable wavelength detector. Results were analyzed using Chromeleon™ 7 software.
HPLC method parameters are provided in Table 11. The method was run 10 minutes for calibration curve samples and 15 minutes for experimental samples.
All solvents and diluents were HPLC grade and all standard dilutions were made using 70:30 10 mM phosphate buffer:ACN. Rebaudioside A standards were obtained from Sigma-Aldrich and stevioside hydrate standards were obtained from Adipo Gen Life Sciences. Stock solutions of 1000 ppm standards were prepared and used to make serial dilutions, as shown in Table 12.
Standard samples of rebA and stevioside were run separately and together in order to determine the order of elution. Areas of chromatogram peaks were determined by software provided by the HPLC manufacturer and used to construct calibration curves. Repeated injections revealed low standard deviations and low variation coefficients. Calibration curves for rebA and stevioside are shown in
Following the development of calibration curves, steviol glycosides were produced and extracted as follows. Yeast transformed independently with SEQ ID NOs. 15 and 16 according to the protocols described above were incubated, separately, in yeast malt medium with 2% raffinose and induction with galactose at 30° C. for 72 hours. Samples were centrifuged at 9,000 rpm for 15 min to produce a pellet. The pellet was resuspended at 1 g/50 mL in sterile deionized water and sonicated 3 times for a total of 2 min, 30 s. Samples were again centrifuged at 9,000 rpm for 15 min and the supernatant was filtered with a 0.45 μm filter. RebA and stevioside were then quantified using HPLC and the calibration curves described above.
HPLC samples were prepared using 1 mL of extracts prepared as described above, brought to a final volume of 5 mL with 70:30 10 mM phosphate buffer:ACN. Samples were analyzed in duplicant. A small shift in retention times for rebA and stevioside was observed in experimental samples as compared to standard samples, with rebA having a peak at 2.514 min and stevioside having a peak at 2.667 min.
Chromatograms of experimental samples extracted from devices having SEQ ID NO. 15 and SEQ ID NO. 16 are shown in
Extracts from the device having SEQ ID NO. 16 showed a higher ratio of rebA to stevioside, indicating a sweeter flavor, since stevioside is responsible for the bitter taste associated with stevia-based sweeteners.
Growth of Device
DNA stevia yeast device (
Extraction of Steviol Glycosides from DNA Device
Extraction of steviol glycosides was achieved by adding lyticase to the culture for 4 hours and then centrifuging the culture at 10,000 RPM for 10 minutes to produce a pellet (6.1 g). The pellet was resuspended in distilled deionized water and sonicated with 4 pulses of 30 seconds each. Sonication was repeated. The solution was then centrifuged again, and the supernatant was filtrated with different pore sizes (i.e. 3, 2, 0.8, 0.6, 0.4 μm), with 0.45 μm the preferred size.
Concentration of Steviol Glycosides
The concentration of steviol glycosides produced by the device was determined using the standard curves based on rebaudioside A or stevioside. Different dilutions solutions (i.e. 6-7) were used and measured using an UV-VIS Spectrophotometer Model Lambda 365 (PerkinElmer CT, USA) set at 203 nm and 210 nm wavelengths. Standard curves based on stevioside and rebaudioside A were used as a reference to determine the concentration of the steviol glycosides present in the extract produced from the device.
The concentration measurement was performed after 10 days growth of the device culture (see above). The steviol glycoside concentration based on stevioside and rebaudioside A was 100 mg/ml and 89 mg/ml, respectively.
Sweetness Determination
The sugar concentration produced by the cultures of yeast transformed with the construct in
Based on the results above, the stevia device produced more sucrose compared to yeast not transformed with the construct based on the increased % sweetness results. Moreover, the yeast transformed with the construct grew quicker and produced a higher population of cells compared to the non-transformed yeast.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions, and methods described herein.
Various modifications and variations can be made to the compounds, compositions, and methods described herein. Other aspects of the compounds, compositions, and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions, and methods disclosed herein. It is intended that the specification and examples be considered as exemplary.
This application claims priority upon U.S. provisional application Serial Nos. 62/557,220 filed on Sep. 12, 2017 and 62/687,284 filed Jun. 20, 2018. These applications are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/050143 | 9/10/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62667284 | May 2018 | US | |
| 62557220 | Sep 2017 | US |
