Patent Application
20200291442
This disclosure relates to recombinant production of steviol glycosides, glycosides of steviol precursors, and steviol glycoside precursors in recombinant hosts. In particular, this disclosure relates to production of steviol glycosides comprising steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (RebI), dulcoside A, mono-glycosylated ent-kaurenoic acids, di-glycosylated ent-kaurenoic acids, tri-glycosylated ent-kaurenoic acids, mono-glycosylated ent-kaurenols, di-glycosylated ent-kaurenols, tri-glycosylated ent-kaurenols, tri-glycosylated steviol glycosides, tetra-glycosylated steviol glycosides, penta-glycosylated steviol glycosides, hexa-glycosylated steviol glycosides, hepta-glycosylated steviol glycosides, or isomers thereof in recombinant hosts.
Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of Stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
Chemical structures for several steviol glycosides are shown in
Recovery and purification of steviol glycosides from the Stevia plant have proven to be labor intensive and inefficient. Moreover, steviol glycoside compositions obtained from a plant-derived Stevia extract generally contain Stevia plant-derived components that can contribute to off-flavors. As such, there remains a need for a recombinant production system that can accumulate high yields of desired steviol glycosides, such as Reb A, RebD, and/or RebM and produce steviol glycoside compositions that are enriched for a one or more desired steviol glycosides relative to a steviol glycoside composition of Stevia plant with a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract. There also remains a need for improved production of steviol glycosides in recombinant hosts for commercial uses. As well, there remains a need for increasing uridine diphosphate glucose (UDP-glucose) formation in recombinant hosts in order to produce higher yields of steviol glycosides, including Reb A, RebD, and/or RebM.
It is against the above background that the present invention provides certain advantages over the prior art.
Although this invention as disclosed herein is not limited to specific advantages or functionalities (such for example, the ability to scale up production of a one or more steviol glycosides or glycosides of a steviol precursor, purify the one or more steviol glycosides or glycosides of the steviol precursor, and produce steviol glycoside compositions where the different proportions of the various steviol glycosides provide the advantage of having a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract), the invention provides a recombinant host cell capable of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture, comprising:
In one aspect of the recombinant host cells disclosed herein, the polypeptide capable of debranching glycogen is capable of 4-α-glucanotransferase activity and α-1,6-amyloglucosidase activity.
In one aspect, the recombinant host cells disclosed herein further comprise:
In one aspect of the recombinant host cells disclosed herein:
In one aspect, the recombinant host cells disclosed herein further comprise:
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cells disclosed herein:
In one aspect, the recombinant host cells disclosed herein comprise:
one or more of:
wherein at least one of the genes is a recombinant gene.
In one aspect, the recombinant host cells disclosed herein comprise:
In one aspect of the recombinant host cells disclosed herein, the gene encoding the polypeptide capable of debranching glycogen and/or the gene encoding the polypeptide capable of synthesizing glucose-1-phosphate are overexpressed by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, expression of the one or more recombinant genes increase the amount of UDP-glucose accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes increases the amount of UDP-glucose accumulated by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes increases an amount of the one or more steviol glycosides or the steviol glycoside composition produced by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes increases the amount of the one or more steviol glycosides produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes increases an amount of RebA, RebD, and/or RebM produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes decreases the amount of the one of one or more steviol glycosides or the steviol glycoside composition accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes decreases the amount of the one or more steviol glycosides accumulated by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell lacking the one or more recombinant genes relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes decreases an amount of 13-SMG accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes increases the amount of total steviol glycosides produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the expression of the one or more recombinant genes decreases the amount of total steviol glycosides produced by the cell by less than 10%, or less than 5%, or less than 2.5% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cells disclosed herein, the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-Stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a Saccharomyces cerevisiae cell.
In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a Yarrowia lipolytica cell.
The invention also provides a method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture, comprising culturing the recombinant host cells disclosed herein in the cell culture, under conditions in which the genes are expressed, and wherein the one or more steviol glycosides or the steviol glycoside composition is produced by the recombinant host cell.
In one aspect of the methods disclosed herein, the genes are constitutively expressed.
In one aspect of the methods disclosed herein, the expression of the genes is induced.
In one aspect of the methods disclosed herein, the amount of RebA, RebD, and/or RebM produced by the cell is increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the methods disclosed herein, the amount of 13-SMG accumulated by the cell is decreased by at least 10%, at least 25%, or at least 50% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the methods disclosed herein, the amount of total steviol glycosides produced by the cell is increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the methods disclosed herein, the amount of total steviol glycosides produced by the cell is decreased by less than 10%, or less than 5%, or less than 2.5% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the methods disclosed herein, the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the one or more steviol glycosides or the steviol glycoside composition.
In one aspect of the methods disclosed herein, the amount of UDP-glucose accumulated by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect, the methods disclosed herein further comprise isolating the produced one or more steviol glycosides or the steviol glycoside composition from the cell culture.
In one aspect of the methods disclosed herein, the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or the steviol glycoside composition, and:
thereby isolating the produced one or more steviol glycosides or the steviol glycoside composition.
In one aspect, the methods disclosed herein further comprise recovering the one or more steviol glycosides or the steviol glycoside composition from the cell culture.
In one aspect of the methods disclosed herein, the recovered one or more steviol glycosides or the steviol glycoside composition is enriched for the one or more steviol glycosides relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.
The invention also provides a method for producing one or more steviol glycosides or a steviol glycoside composition, comprising whole-cell bioconversion of a plant-derived or synthetic steviol and/or steviol glycosides in a cell culture of a recombinant host cell using:
optionally, one or more of:
one or more of:
wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides or the steviol glycoside composition thereby.
In one aspect of the methods disclosed herein:
In one aspect of the methods disclosed herein, the recombinant host cell is a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
In one aspect of the methods disclosed herein, the recombinant host cell is a Saccharomyces cerevisiae cell.
In one aspect of the methods disclosed herein, the recombinant host cell is a Yarrowia lipolytica cell.
In one aspect of the methods disclosed herein, the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.
The invention also provides a cell culture, comprising the recombinant host cells disclosed herein, the cell culture further comprising:
wherein the one or more steviol glycosides or the steviol glycoside composition is present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more steviol glycosides or the steviol glycoside composition relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a cell culture, comprising the recombinant host cells disclosed herein, the cell culture further comprising:
wherein UDP-glucose is present in the cell culture at a concentration of at least 100 μM;
wherein the cell culture is enriched for UGP-glucose relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a cell lysate from the recombinant host cells disclosed herein grown in the cell culture, comprising:
wherein the one or more steviol glycosides or the steviol glycoside composition produced by the recombinant host cell is present at a concentration of at least 1 mg/liter of the cell culture.
The invention also provides one or more steviol glycosides produced by the recombinant host cells disclosed herein;
wherein the one or more steviol glycosides produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides one or more steviol glycosides produced by the methods disclosed herein;
wherein the one or more steviol glycosides produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a sweetener composition, comprising the one or more steviol glycosides disclosed herein.
The invention also provides a food product comprising, the sweetener composition disclosed herein.
The invention also provides a beverage or a beverage concentrate, comprising the sweetener composition disclosed herein.
These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids.
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, Calif.).
As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
As used herein, the terms “microorganism,” “microorganism host,” and “microorganism host cell” can be used interchangeably. As used herein, the terms “recombinant host” and “recombinant host cell” can be used interchangeably. The person of ordinary skill in the art will appreciate that the terms “microorganism,” microorganism host,” and “microorganism host cell,” when used to describe a cell comprising a recombinant gene, may be taken to mean “recombinant host” or “recombinant host cell.” As used herein, the term “recombinant host” is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. In some aspects, the introduced DNA is introduced into the genome in a location different than where the corresponding endogenous DNA segment originally resided. Suitable recombinant hosts include microorganisms.
As used herein, the term “recombinant gene” refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
As used herein, the term “engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
As used herein, the term “endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841-54. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65. In some aspects, overexpression can be performed by integration using the USER cloning system; see, e.g., Nour-Eldin et al., 2010, Methods Mol Biol. 643:185-200. As used herein, the terms “deletion,” “deleted,” “knockout,” and “knocked out” can be used interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae. In some aspects, the terms “deletion,” “deleted,” “knockout,” and “knocked out” can be used interchangeably to refer to an endogenous gene that has been mutated so that the endogenous gene has reduced activity or no activity.
As used herein, the terms “heterologous sequence” and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
As used herein, the terms “heterologous sequence” and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
As used herein, the term “constitutive,” “constitutive expression,” or “constitutively expressed” refers to a continuous transcription of a gene resulting in the continuous expression of a protein.
As used herein, the term “inducible,” “inducible expression,” or “inducibly expressed” refers to the expression of a gene in response to a stimuli. Stimuli include, but are not limited to, chemicals, stress, or biotic stimuli.
A “selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
As used herein, the terms “variant” and “mutant” are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
As used herein, the term “inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
As used herein, the term “steviol glycoside” refers to rebaudioside A (RebA) (CAS #58543-16-1), rebaudioside B (RebB) (CAS #58543-17-2), rebaudioside C (RebC) (CAS #63550-99-2), rebaudioside D (RebD) (CAS #63279-13-0), rebaudioside E (RebE) (CAS #63279-14-1), rebaudioside F (RebF) (CAS #438045-89-7), rebaudioside M (RebM) (CAS #1220616-44-3), Rubusoside (CAS #63849-39-4), Dulcoside A (CAS #64432-06-0), rebaudioside I (RebI) (MassBank Record: FU000332), rebaudioside Q (RebQ), 1,2-Stevioside (CAS #57817-89-7), 1,3-Stevioside (RebG), Steviol-1,2-Bioside (MassBank Record: FU000299), Steviol-1,3-Bioside, Steviol-13-O-glucoside (13-SMG), Steviol-19-O-glucoside (19-SMG), a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and isomers thereof. See
As used herein, the terms “steviol glycoside precursor” and “steviol glycoside precursor compound” are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway. Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), ent-copalyl-diphosphate, ent-kaurene, ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, and steviol. See
As used herein, the term “contact” is used to refer to any physical interaction between two objects. For example, the term “contact” may refer to the interaction between an enzyme and a substrate. In another example, the term “contact” may refer to the interaction between a liquid (e.g., a supernatant) and an adsorbent resin.
Steviol glycosides and/or steviol glycoside precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion. As used herein, the terms “produce” and “accumulate” can be used interchangeably to describe synthesis of steviol glycosides and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
As used herein, the terms “culture broth,” “culture medium,” and “growth medium” can be used interchangeably to refer to a liquid or solid that supports growth of a cell. A culture broth can comprise glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids. The trace metals can be divalent cations, including, but not limited to, Mn2+ and/or Mg2+. In some embodiments, Mn2+ can be in the form of MnCl2 dihydrate and range from approximately 0.01 g/L to 100 g/L. In some embodiments, Mg2+ can be in the form of MgSO4 heptahydrate and range from approximately 0.01 g/L to 100 g/L. For example, a culture broth can comprise i) approximately 0.02-0.03 g/L MnCl2 dihydrate and approximately 0.5-3.8 g/L MgSO4 heptahydrate, ii) approximately 0.03-0.06 g/L MnCl2 dihydrate and approximately 0.5-3.8 g/L MgSO4 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCl2 dihydrate and approximately 0.5-7.3 g/L MgSO4 heptahydrate. Additionally, a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety. Methods of producing steviol glycosides in recombinant hosts, by whole cell bio-conversion, and in vitro are also described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) (e.g., a geranylgeranyl diphosphate synthase (GGPPS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-copalyldiphosphate from GGPP (e.g., a ent-copalyl diphosphate synthase (CDPS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a kaurene synthase (KS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a kaurene oxidase (KO) polypeptide); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a cytochrome P450 reductase (CPR) polypeptide or a P450 oxidoreductase (POR) polypeptide; for example, but not limited to a polypeptide capable of electron transfer from NADPH to cytochrome P450 complex during conversion of NADPH to NADP+, which is utilized as a cofactor for terpenoid biosynthesis); a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a steviol synthase (KAH) polypeptide); and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., an ent-copalyl diphosphate synthase (CDPS)—ent-kaurene synthase (KS) polypeptide) can produce steviol in vivo. See, e.g.,
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a UGT85C2 polypeptide); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT76G1 polypeptide); a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group (e.g., a UGT74G1 polypeptide); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT91D2 or a EUGT11 polypeptide) can produce a steviol glycoside in vivo. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo. See, e.g.,
In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside polypeptides.
In some aspects, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a polypeptide capable of glycosylating steviol or the steviol glycoside at its C-19 carboxyl group, and/or a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, transfers a glucose molecule from uridine diphosphate glucose (UDP-glucose) to steviol and/or a steviol glycoside.
In some aspects, UDP-glucose is produced in vivo through expression of one or more enzymes involved in the UDP-glucose biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of transporting uracil into the host cell (e.g., uracil permease (FUR4)); a gene encoding a polypeptide capable of synthesizing uridine monophosphate (UMP) from uracil (e.g., uracil phosphoribosyltransferase (FUR1)); a gene encoding a polypeptide capable of synthesizing orotidine monophosphate (OMP) from orotate or orotic acid (e.g., orotate phosphoribosyltransferase 1 (URA5) and orotate phosphoribosyltransferase 2 (URA10)); a gene encoding a polypeptide capable of synthesizing UMP from OMP (e.g., orotidine 5′-phosphate decarboxylase (URA3)); a gene encoding a polypeptide capable of synthesizing uridine diphosphate (UDP) from UMP (e.g., uridylate kinase (URA6)); a gene encoding a polypeptide capable of synthesizing uridine 5′-triphosphate (UTP) from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates, e.g., nucleoside diphosphate kinase (YNK1)); a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., phosphoglucomutase-1 (PGM1) and phosphoglucomutase-2 (PGM2)); a gene encoding a polypeptide capable of debranching glycogen (e.g., glycogen debranching enzyme (GDB1)); a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., glycogen phosphorylase (GPH1)); and/or a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., UTP-glucose-1-phosphate uridylyltransferase (UGP1)) can produce UDP-glucose in vivo. See, e.g.,
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of synthesizing UTP from UDP. In some aspects, the gene encoding a polypeptide capable of synthesizing UTP from UDP is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator, for example but not limited to, tCYC1 (SEQ ID NO:154) or tADH1 (SEQ ID NO:155). In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter, for example but not limited to, pTEF1 (SEQ ID NO:148), pPGK1 (SEQ ID NO:149), pTDH3 (SEQ ID NO:150), pTEF2 (SEQ ID NO:151), pTPI1 (SEQ ID NO:152), or pPDC1 (SEQ ID NO:153). In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP results in a total expression level of genes encoding a polypeptide capable of synthesizing UTP from UDP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UTP from UDP, i.e., an overexpression of a polypeptide capable of synthesizing UTP from UDP.
In some aspects, the gene encoding the polypeptide capable of synthesizing UTP from UDP is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the polypeptide capable of synthesizing UTP from UDP can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a polypeptide capable of synthesizing UTP from UDP (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
The person of ordinary skill in the art will appreciate that, e.g., expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP; expression of a recombinant gene and an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP, and expression of an endogenous gene encoding a polypeptide capable of synthesizing UTP from UDP, wherein the wild-type promoter and/or enhancer of the endogenous gene are exchanged for a strong promoter and/or enhancer, each result in overexpression of a polypeptide capable of synthesizing UTP from UDP relative to a corresponding host not expressing a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP and/or a corresponding host expressing only a native gene encoding a polypeptide capable of synthesizing UTP from UDP, operably linked to the wild-type promoter and enhancer—i.e., as used herein, the term “expression” may include “overexpression.”
In some embodiments, a polypeptide capable of synthesizing UTP from UDP is overexpressed such that the total expression level of genes encoding the polypeptide capable of synthesizing UTP from UDP is at least 5% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UTP from UDP. In some embodiments, the total expression level of genes encoding a polypeptide capable of synthesizing UTP from UDP is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UTP from UDP.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate. In some aspects, the gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator, for example but not limited to, tCYC1 (SEQ ID NO:154) or tADH1 (SEQ ID NO:155). In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter, for example but not limited to, pTEF1 (SEQ ID NO:148), pPGK1 (SEQ ID NO:149), pTDH3 (SEQ ID NO:150), pTEF2 (SEQ ID NO:151), pTPI1 (SEQ ID NO:152), or pPDC1 (SEQ ID NO:153). In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate results in a total expression level of genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate that is higher than the expression level of endogenous genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, i.e., an overexpression of a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate.
In some aspects, the gene encoding the polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
In some embodiments, a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate is overexpressed such that the total expression level of genes encoding the polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate is at least 5% higher than the expression level of endogenous genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate. In some embodiments, the total expression level of genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of debranching glycogen. In some aspects, debranching glycogen comprises glycogen breakdown and/or glucose mobilization. In some aspects, debranching glycogen comprises breakdown of glycogen into glucose-1-phosphate. In some aspects, the polypeptide capable of debranching glycogen comprises a polypeptide capable of intramolecularly transferring α-1,4-linked glucose and/or α-1,4-linked glucan of glycogen to a new position (i.e., 4-α-glucanotransferase activity), and/or capable of hydrolyzing an α-1,6 linkage of glycogen (i.e., α-1,6-amyloglucosidase activity). In some aspects, the polypeptide capable of debranching glycogen comprises a bifunctional polypeptide capable of 4-α-glucanotransferase activity and capable of α-1,6-amyloglucosidase activity. In some aspects, the recombinant host can comprise a first polypeptide capable of 4-α-glucanotransferase activity and a second peptide capable of α-1,6-amyloglucosidase activity. In some aspects, the gene encoding a polypeptide capable of debranching glycogen is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator, for example but not limited to, tCYC1 (SEQ ID NO:154) or tADH1 (SEQ ID NO:155). In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter, for example but not limited to, pTEF1 (SEQ ID NO:148), pPGK1 (SEQ ID NO:149), pTDH3 (SEQ ID NO:150), pTEF2 (SEQ ID NO:151), pTPI1 (SEQ ID NO:152), or pPDC1 (SEQ ID NO:153). In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen results in a total expression level of genes encoding a polypeptide capable of debranching glycogen that is higher than the expression level of endogenous genes encoding a polypeptide capable of debranching glycogen, i.e., an overexpression of a polypeptide capable of debranching glycogen.
In some aspects, the gene encoding the polypeptide capable of debranching glycogen is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the polypeptide capable of debranching glycogen can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a polypeptide capable of debranching glycogen can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a polypeptide capable of debranching glycogen (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of debranching glycogen, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of debranching glycogen, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a polypeptide capable of debranching glycogen, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
In some embodiments, a polypeptide capable of debranching glycogen is overexpressed such that the total expression level of genes encoding the polypeptide capable of debranching glycogen is at least 5% higher than the expression level of endogenous genes encoding a polypeptide capable of debranching glycogen. In some embodiments, the total expression level of genes encoding a polypeptide capable of debranching glycogen is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a polypeptide capable of debranching glycogen.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen. In some aspects, the gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen comprises a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and an α-1,4-linked glucose of glycogen. In some aspects, the gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator, for example but not limited to, tCYC1 (SEQ ID NO:154) or tADH1 (SEQ ID NO:155). In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter, for example but not limited to, pTEF1 (SEQ ID NO:148), pPGK1 (SEQ ID NO:149), pTDH3 (SEQ ID NO:150), pTEF2 (SEQ ID NO:151), pTPI1 (SEQ ID NO:152), or pPDC1 (SEQ ID NO:153). In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen results in a total expression level of genes encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, i.e., an overexpression of a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen.
In some aspects, the gene encoding the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
In some embodiments, a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen is overexpressed such that the total expression level of genes encoding the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen is at least 5% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen. In some embodiments, the total expression level of genes encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some aspects, the gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator, for example but not limited to, tCYC1 (SEQ ID NO:154) or tADH1 (SEQ ID NO:155). In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter, for example but not limited to, pTEF1 (SEQ ID NO:148), pPGK1 (SEQ ID NO:149), pTDH3 (SEQ ID NO:150), pTEF2 (SEQ ID NO:151), pTPI1 (SEQ ID NO:152), or pPDC1 (SEQ ID NO:153). In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate results in a total expression level of genes encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, i.e., an overexpression of a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
In some aspects, the gene encoding the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
In some embodiments, a recombinant host comprising a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate is overexpressed such that the total expression level of genes encoding the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate is at least 5% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some embodiments, the total expression level of genes encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
In some aspects, a recombinant host comprising one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP, one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate, one or more genes encoding one or more polypeptide capable of debranching glycogen, one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate may further comprise a recombinant gene encoding a polypeptide capable of transporting uracil into the host cell; a recombinant gene encoding a polypeptide capable of synthesizing uridine monophosphate (UMP) from uracil; a recombinant gene encoding a polypeptide capable of synthesizing orotidine monophosphate (OMP) from orotate or orotic acid; a recombinant gene encoding a polypeptide capable of synthesizing UMP from OMP; and/or a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate (UDP) from UMP. In some embodiments, a recombinant host comprising one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP, one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate, one or more genes encoding one or more polypeptides capable of debranching glycogen, one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate may overexpress a gene encoding a polypeptide capable of transporting uracil into the host cell; a gene encoding a polypeptide capable of synthesizing uridine monophosphate (UMP) from uracil; a gene encoding a polypeptide capable of synthesizing orotidine monophosphate (OMP) from orotate or orotic acid; a gene encoding a polypeptide capable of synthesizing UMP from OMP; and/or a gene encoding a polypeptide capable of synthesizing uridine diphosphate (UDP) from UMP.
In some aspects, the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:123 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:122).
In some aspects, the polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:1), SEQ ID NO:119 (encoded by the nucleotide sequence set forth in SEQ ID NO:118), SEQ ID NO:141 (encoded by the nucleotide sequence set forth in SEQ ID NO:140), SEQ ID NO:143 (encoded by the nucleotide sequence set forth in SEQ ID NO:142), SEQ ID NO:145 (encoded by the nucleotide sequence set forth in SEQ ID NO:144), or SEQ ID NO:147 (encoded by the nucleotide sequence set forth in SEQ ID NO:146).
In some aspects, the polypeptide capable of debranching glycogen comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:157 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:156).
In some aspects, the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:159 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:158).
In some aspects, the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:120), SEQ ID NO:125 (encoded by the nucleotide sequence set forth in SEQ ID NO:124), SEQ ID NO:127 (encoded by the nucleotide sequence set forth in SEQ ID NO:126), SEQ ID NO:129 (encoded by the nucleotide sequence set forth in SEQ ID NO:128), SEQ ID NO:131 (encoded by the nucleotide sequence set forth in SEQ ID NO:130), SEQ ID NO:133 (encoded by the nucleotide sequence set forth in SEQ ID NO:132), SEQ ID NO:135 (encoded by the nucleotide sequence set forth in SEQ ID NO:134), SEQ ID NO:137 (encoded by the nucleotide sequence set forth in SEQ ID NO:136), or SEQ ID NO:139 (encoded by the nucleotide sequence set forth in SEQ ID NO:138).
In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP and a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of debranching glycogen and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, a polypeptide capable of synthesizing UTP from UDP, and a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate. In some embodiments, a recombinant host comprises a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
In some embodiments, a recombinant host comprises two or more recombinant genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway, e.g., a gene encoding a polypeptide capable of converting glucose-6-phosphate having a first amino acid sequence and a gene encoding a polypeptide capable of converting glucose-6-phosphate having a second amino acid sequence distinct from the first amino acid sequence. For example, in some embodiments, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence of PGM1 (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2) and a gene encoding a polypeptide having the amino acid sequence of PGM2 (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:147). In certain such embodiments, the two or more genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway comprise nucleotide sequences native to the recombinant host cell (e.g., a recombinant S. cerevisiae host cell comprising a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:119). In other such embodiments, one of the two or more genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway comprises a nucleotide sequence native to the recombinant host cell, while one or more of the two or more genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway comprises a heterologous nucleotide sequence. For example, in some embodiments, a recombinant S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121 (i.e., a recombinant host overexpressing the polypeptide) further expresses a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in, e.g., SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139. In another example, in some embodiments, a recombinant S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:119 (i.e., a recombinant host overexpressing the polypeptide) further expresses a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in, e.g., SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:147. Accordingly, as used herein, the term “a recombinant gene” may include “one or more recombinant genes.”
In some embodiments, a recombinant host comprises two or more copies of a recombinant gene encoding a polypeptide involved in the UDP-glucose biosynthetic pathway or the steviol glycoside biosynthetic pathway. In some embodiments, a recombinant host is preferably transformed with, e.g., two copies, three copies, four copies, or five copies of a recombinant gene encoding a polypeptide involved in the UDP-glucose biosynthetic pathway or the steviol glycoside biosynthetic pathway. For example, in some embodiments, a recombinant host is transformed with two copies of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), two copies of a recombinant gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), or two copies of a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159). The person of ordinary skill in the art will appreciate that, in some embodiments, recombinant genes may be replicated in a host cell independently of cell replication; accordingly, a recombinant host cell may comprise, e.g., more copies of a recombinant gene than the number of copies the cell was transformed with. Accordingly, as used herein, the term “a recombinant gene” may include “one or more copies of a recombinant gene.”
In some aspects, expression of a polypeptide capable of synthesizing UTP from UDP, a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a polypeptide capable of debranching glycogen, a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a recombinant host cell increases the amount of UDP-glucose produced by the cell. In some aspects, expression of a polypeptide capable of synthesizing UTP from UDP, a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a polypeptide capable of debranching glycogen, a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a recombinant host cell maintains, or even increases, the pool of UDP-glucose available for, e.g., glycosylation of a steviol or a steviol glycoside. In some aspects, expression of a polypeptide capable of synthesizing UTP from UDP, a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a polypeptide capable of debranching glycogen, a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a recombinant host cell increases the speed with which UDP-glucose is regenerated, thus maintaining, or even increasing, the UDP-glucose pool, which can be used to synthesize one or more steviol glycosides.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157) and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159) in a recombinant host cell increases the amount of UDP-glucose produced by the cell by at least 10%, e.g., at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200%, or at least 225%, or at least 250%, or at least 275%, or at least 300%, calculated as an increase in intracellular UDP-glucose concentration relative to a corresponding host lacking the recombinant genes.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g. a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:147), a recombinant gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139) in a recombinant host cell increases the amount of UDP-glucose produced by the cell by at least 10%, e.g., at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200%, or at least 225%, or at least 250%, or at least 275%, or at least 300%, calculated as an increase in intracellular UDP-glucose concentration relative to a corresponding host lacking the recombinant genes.
In certain such embodiments, one or more of the recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, the recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, the recombinant gene encoding a polypeptide capable of debranching glycogen, the recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and the recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate comprise a nucleotide sequence native to the host cell. For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157 and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159 in a steviol glycoside-producing S. cerevisiae host cell (i.e., providing a recombinant host overexpressing the polypeptides) increases the amount of UDP-glucose produced by the cell by at least 10%, e.g., at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200%, or at least 225%, or at least 250%, or at least 275%, or at least 300%, calculated as an increase in intracellular UDP-glucose concentration relative to a corresponding host lacking the recombinant genes.
In another example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP having the amino acid sequence set forth in SEQ ID NO:123, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:2 and/or SEQ ID NO:119, a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121 in a steviol glycoside-producing S. cerevisiae host cell (i.e., providing a recombinant host overexpressing the polypeptides) increases the amount of UDP-glucose produced by the cell by at least 10%, e.g., at least 25%, or at least 50%, or at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200%, or at least 225%, or at least 250%, or at least 275%, or at least 300%, calculated as an increase in intracellular UDP-glucose concentration relative to a corresponding host lacking the recombinant genes.
In some aspects, expression of a polypeptide capable of synthesizing UTP from UDP, a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a polypeptide capable of debranching glycogen, a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a steviol-glycoside producing recombinant host cell further expressing a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, increases the amount of one or more steviol glycosides produced by the cell, and/or decreases the amount of one or more steviol glycosides produced by the cell. In some embodiments, the steviol glycoside-producing host further expresses a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; and a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyldiphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate.
In some aspects, the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:19), SEQ ID NO:22 (encoded by the nucleotide sequence set forth in SEQ ID NO:21), SEQ ID NO:24 (encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (encoded by the nucleotide sequence set forth in SEQ ID NO:31), or SEQ ID NO:116 (encoded by the nucleotide sequence set forth in SEQ ID NO:115). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (encoded by the nucleotide sequence set forth in SEQ ID NO:41). In some embodiments, the polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP lacks a chloroplast transit peptide. In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (encoded by the nucleotide sequence set forth in SEQ ID NO:51). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some embodiments, a recombinant host comprises a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate. In some aspects, the bifunctional polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:54 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:53), SEQ ID NO:56 (encoded by the nucleotide sequence set forth in SEQ ID NO:55), or SEQ ID NO:58 (encoded by the nucleotide sequence set forth in SEQ ID NO:57). In some embodiments, a recombinant host comprising a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (encoded by the nucleotide sequence set forth in SEQ ID NO:61), SEQ ID NO:117 (encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (encoded by the nucleotide sequence set forth in SEQ ID NO:71), SEQ ID NO:74 (encoded by the nucleotide sequence set forth in SEQ ID NO:73), or SEQ ID NO:76 (encoded by the nucleotide sequence set forth in SEQ ID NO:75). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:77), SEQ ID NO:80 (encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (encoded by the nucleotide sequence set forth in SEQ ID NO:81), SEQ ID NO:84 (encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (encoded by the nucleotide sequence set forth in SEQ ID NO:91). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of reducing cytochrome P450 complex further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of synthesizing steviol from ent-kaurenoic acid comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO:100 (encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:106 (encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (encoded by the nucleotide sequence set forth in SEQ ID NO:107), SEQ ID NO:110 (encoded by the nucleotide sequence set forth in SEQ ID NO:109), SEQ ID NO:112 (encoded by the nucleotide sequence set forth in SEQ ID NO:111), or SEQ ID NO:114 (encoded by the nucleotide sequence set forth in SEQ ID NO:113). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some embodiments, a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., UGT85C2 polypeptide) (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., UGT76G1 polypeptide) (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group (e.g., UGT74G1 polypeptide) (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., EUGT11 polypeptide) (SEQ ID NO:16). In some aspects, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., UGT91D2 polypeptide) can be a UGT91D2e polypeptide (SEQ ID NO:11) or a UGT91D2e-b polypeptide (SEQ ID NO:13). In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside further comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139). In some embodiments, the recombinant host is an S. cerevisiae host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In some aspects, the polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6, the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:8, the polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group is encoded by the nucleotide sequence set forth in SEQ ID NO:3, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:15. The skilled worker will appreciate that expression of these genes may be necessary to produce a particular steviol glycoside but that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen in a steviol glycoside-producing recombinant host increases the amount of one or more steviol glycosides, e.g., RebA, RebD, and/or RebM, produced by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding host lacking the one or more recombinant genes.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157) and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159) in a steviol glycoside-producing host increases the amount of one or more steviol glycosides, e.g., RebA, RebD, and/or RebM, produced by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding host lacking the one or more recombinant genes.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a steviol glycoside-producing recombinant host increases the amount of one or more steviol glycosides, e.g., rubusoside, RebB, RebA, RebD, and/or RebM, produced by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding host lacking the one or more recombinant genes.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g. a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, or SEQ ID NO:147), a recombinant gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139) in a steviol glycoside-producing host increases the amount of one or more steviol glycosides, e.g., rubusoside, RebB, RebA, RebD, and/or RebM, produced by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding host lacking the one or more recombinant genes.
In some embodiments, expression of a recombinant gene encoding a recombinant gene encoding a polypeptide capable of debranching glycogen and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen in a steviol glycoside-producing recombinant host decreases the amount of one or more steviol glycosides, e.g., 13-SMG, produced by the cell by at least 5%, e.g., at least 10%, or at least 15%, or at least 20%, or at least 25%, calculated as a decrease in intracellular steviol glycoside concentration relative to a corresponding steviol glycoside-producing host lacking the recombinant genes.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157 and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159 in a steviol glycoside-producing recombinant host decreases the amount of 13-SMG produced by the cell by at least 5%, e.g., at least 7.5%, or at least 10%, or at least 15%, or at least 20%, at least 25%, or at least 50%, calculated as decrease in intracellular 13-SMG concentration relative to a corresponding host lacking the one or more recombinant genes.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a steviol glycoside-producing recombinant host decreases the amount of one or more steviol glycosides, e.g., 13-SMG and RebD, produced by the cell by at least 5%, e.g., at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, calculated as a decrease in intracellular steviol glycoside concentration relative to a corresponding steviol glycoside-producing host lacking the recombinant genes.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP having the amino acid sequence set forth in SEQ ID NO:123, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:2, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:119, a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159, a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121, and further expression of a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in, e.g., SEQ ID NO:127, SEQ ID NO:133, SEQ ID NO:129, SEQ ID NO:125, SEQ ID NO:139, or SEQ ID NO:135, in a steviol glycoside-producing recombinant host decreases the amount of 13-SMG produced by the cell by at least 5%, e.g., at least 7.5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, at least 35%, or at least 50%, calculated as a decrease in intracellular 13-SMG concentration relative to a corresponding host lacking the one or more recombinant genes.
In some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides (i.e., the total amount of mono-, di-, tri-, tetra-penta-, hexa-, and hepta-glycosylated steviol compounds) by at least 5%, e.g., at least 7.5%, or at least 10%, or at least 12.5%, or at least 15%, or at least 17.5%, or at least 20%, or at least 25%, or at least 27.5%, or at least 30%, or at least 35%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding steviol glycoside-producing host lacking the recombinant genes.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP having the amino acid sequence set forth in SEQ ID NO:123, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:2, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:119, a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159, a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121, and further expression of a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in, e.g., SEQ ID NO:133, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:125, SEQ ID NO:139, or SEQ ID NO:135, in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides (i.e., the total amount of mono-, di-, tri-, tetra-penta-, hexa-, and hepta-glycosylated steviol compounds) by at least 5%, e.g., at least 7.5%, or at least 10%, or at least 12.5%, or at least 15%, or at least 17.5%, or at least 20%, or at least 25%, or at least 27.5%, or at least 30%, or at least 35%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding steviol glycoside-producing host lacking the recombinant genes.
In some other embodiments, the total amount of steviol glycosides produced by a steviol glycoside-producing recombinant host cell is unchanged (i.e., increased or decreased by less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%) by expression in the host of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a recombinant gene encoding a polypeptide capable of debranching glycogen, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157 and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159 in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides produced by the host by less than 5%, e.g., less than 4%, or less than 3%, or less than 2%.
In another example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP having the amino acid sequence set forth in SEQ ID NO:123, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:2, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:119, a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121 in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides produced by the host by less than 5%, e.g., less than 4%, or less than 3%, or less than 2%.
The person of ordinary skill in the art will appreciate that, in such embodiments, expression of one or more genes encoding a polypeptide involved in the involved in the UDP-glucose biosynthetic pathway may affect the relative levels of steviol glycosides produced by the recombinant host, e.g., by increasing the level of UDP-glucose available as a substrate for a polypeptide capable of glycosylating a steviol or a steviol glycoside.
For example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157 and a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159 in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides produced by the host by less than 5%, e.g., less than 4%, or less than 3%, or less than 2%, increases the amount of RebA, RebD, and/or RebM produced by the host by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular steviol glycoside concentration relative to a corresponding host lacking the one or more recombinant genes and decreases the amount of 13-SMG produced by the host cell by at least 5%, e.g., at least 10%, at least 20%, at least 25%, or at least 50%, calculated as a decrease in intracellular 13-SMG concentration relative to a corresponding host lacking the one or more recombinant genes.
In another example, in some embodiments, expression of a recombinant gene encoding a polypeptide capable of synthesizing UTP from UDP having the amino acid sequence set forth in SEQ ID NO:123, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:2, a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:119, a recombinant gene encoding a polypeptide capable of debranching glycogen having the amino acid sequence set forth in SEQ ID NO:157, a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having the amino acid sequence set forth in SEQ ID NO:159, and a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having the amino acid sequence set forth in SEQ ID NO:121 in a steviol glycoside-producing recombinant host increases the total amount of steviol glycosides produced by the host by less than 5%, e.g., less than 4%, or less than 3%, or less than 2%, increases the amount of RebM produced by the host by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250%, calculated as an increase in intracellular RebM concentration relative to a corresponding host lacking the one or more recombinant genes, and decreases the amount of RebD produced by the host by at least 10%, e.g., at least 20%, or at least 30%, at least 40%, or at least 50%, calculated as a decrease in intracellular RebD concentration relative to a corresponding host lacking the one or more recombinant genes.
In some embodiments, a recombinant host cell comprises one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157) and/or one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159). In some embodiments, a recombinant host cell comprises one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139).
In certain embodiments, a recombinant host comprises one or more recombinant genes having a nucleotide sequence native to the host that encode one or more polypeptides capable of synthesizing UTP from UDP, one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate, one or more polypeptides capable of debranching glycogen, one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, i.e., a recombinant host overexpresses one or more polypeptides capable of synthesizing UTP from UDP, one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate, one or more polypeptides capable of debranching glycogen, one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate.
In certain such embodiments, a recombinant host cell overexpresses one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., an S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., an S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, and/or SEQ ID NO:119), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., an S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., an S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., an S. cerevisiae host cell expressing a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121).
In one example, a recombinant S. cerevisiae host cell overexpresses a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:157 and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:159. In another example, a recombinant S. cerevisiae host cell overexpresses a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:123, a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:119, a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:157, and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:159.
In certain embodiments, a recombinant host cell comprising one or more genes encoding one or more polypeptides capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), one or more genes encoding one or more polypeptides capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), one or more genes encoding one or more polypeptides capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139), further comprises a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, a recombinant host comprises two or more genes encoding two or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), and/or two or more genes encoding two or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139).
In certain such embodiments, a recombinant host comprises two or more genes encoding two or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate, e.g., two or more genes encoding two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147. In one example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 and a polypeptide having the amino acid sequence set forth in SEQ ID NO:119. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a polypeptide having the amino acid sequence set forth in SEQ ID NO:119, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:145. In some embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), a gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139).
In certain such embodiments, a recombinant host comprises two or more genes encoding two or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, e.g., two or more genes encoding two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139. In one example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a polypeptide having the amino acid sequence set forth in SEQ ID NO:125. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a polypeptide having the amino acid sequence set forth in SEQ ID NO:127. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a polypeptide having the amino acid sequence set forth in SEQ ID NO:129. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a polypeptide having the amino acid sequence set forth in SEQ ID NO:131. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a gene encoding a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:133. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:135. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:137. In another example, a recombinant host comprises a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:121 and a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:139. In some embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), a gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), and/or one or more genes encoding one or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., one or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147).
In certain such embodiments, a recombinant host comprising two or more genes encoding two or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), and/or two or more genes encoding two or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139) is a host cell overexpressing one or more genes encoding one or more polypeptides involved in the UDP-glucose biosynthetic pathway (e.g., an S. cerevisiae host cell expressing one or more genes encoding one or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:157, and/or SEQ ID NO:159).
In certain embodiments, a recombinant host cell comprising two or more genes encoding two or more polypeptides capable of converting glucose-6-phosphate to glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:119, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, and/or SEQ ID NO:147), and/or two or more genes encoding two or more polypeptides capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., two or more polypeptides having the amino acid sequence set forth in SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, and/or SEQ ID NO:139), further comprises a gene encoding polypeptide capable of synthesizing UTP from UDP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:123), a gene encoding a polypeptide capable of debranching glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:157), a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:159), a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, one or more steviol glycosides or a steviol glycoside composition is produced in an in vitro method, comprising adding a polypeptide capable of debranching glycogen comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157 and/or a polypeptide capable of synthesizing glucose-1-phosphate comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; and, optionally, one or more of: a polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:123; a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:2, 119, or 143 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:141, 145, or 147; and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:121 or 127, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:125, 129, 133, 135, 137, or 139 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:131; and one or more of: a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; and a plant-derived or synthetic steviol, steviol precursors, and/or steviol glycosides to a reaction mixture; wherein at least one of the polypeptide is a recombinant polypeptide; and producing the one or more steviol glycosides or the steviol glycoside composition thereby.
In one aspect of the in vitro methods disclosed herein, the reaction mixture comprises: (a) one or more steviol glycosides or steviol glycoside composition; (b) a polypeptide capable of debranching glycogen having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157 and/or a polypeptide capable of synthesizing glucose-1-phosphate comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; and, optionally, one or more of: a polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:123; a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:2, 119, or 143 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:141, 145, or 147; and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:121 or 127, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:125, 129, 133, 135, 137, or 139 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:131; and one or more of: a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; (c) uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine ; and/or (d) reaction buffer and/or salts.
In one aspect of the in vitro methods disclosed herein, the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.
In some embodiments, one or more steviol glycosides or a steviol glycoside composition is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium. For example, a host cell expressing a gene encoding a polypeptide capable of synthesizing UTP from UDP, a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a gene encoding a polypeptide capable of debranching glycogen, a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate; and further expressing a gene encoding a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium. In certain such embodiments, the host cell may further express a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate.
In some embodiments, the method for producing one or more steviol glycosides or a steviol glycoside composition disclosed herein comprises whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using: (a) a polypeptide capable of debranching glycogen, and/or (b) a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen; optionally, one or more of: (c) a polypeptide capable of synthesizing UTP from UDP, (d) a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and/or (e) a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate; and one or more of: (f) a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof; (g) a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; (h) a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof; and/or (i) a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides or the steviol glycoside composition thereby.
In some embodiments of the methods for producing one or more steviol glycosides or a steviol glycoside composition disclosed herein comprises whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell disclosed herein, the polypeptide capable of debranching glycogen comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:157; and/or the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:159.
In some embodiments, a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-13 hydroxyl group thereof; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof; and/or a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can be displayed on the surface of the recombinant host cells disclosed herein by fusing it with anchoring motifs.
In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
In some embodiments, steviol, one or more steviol glycoside precursors, one or more steviol glycosides, or a steviol glycoside composition are produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the steviol glycoside pathway, produce steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides. For example, a host expressing a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate and a host expressing a gene encoding a polypeptide capable of synthesizing UTP from UDP, a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, a gene encoding a polypeptide capable of debranching glycogen, a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, and/or a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate; and further expressing a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, produce one or more steviol glycosides.
In some embodiments, the steviol glycoside comprises, for example, but not limited to, 13-SMG, steviol-1,2-bioside, steviol-1,3-bioside, 19-SMG, 1,2-stevioside, 1,3-stevioside (RebG), rubusoside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, RebI, dulcoside A, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.
In some embodiments, a steviol glycoside or steviol glycoside precursor composition produced in vivo, in vitro, or by whole cell bioconversion does not comprise or comprises a reduced amount or reduced level of plant-derived components than a Stevia extract from, inter alia, a Stevia plant. Plant-derived components can contribute to off-flavors and include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes, decanoic acid, 8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, β-sitosterol, α- and β-amyrin, lupeol, β-amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellin. In some embodiments, the plant-derived components referred to herein are non-glycoside compounds.
As used herein, the terms “detectable amount,” “detectable concentration,” “measurable amount,” and “measurable concentration” refer to a level of steviol glycosides measured in AUC, μM/OD600, mg/L, μM, or mM. Steviol glycoside production (i.e., total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
As used herein, the term “undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an “undetectable concentration” is not present in a steviol glycoside or steviol glycoside precursor composition.
After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside, steviol and/or one or more steviol glycosides can then be recovered from the culture using various techniques known in the art. Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4° C. to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography. The fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt; and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent.
The supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic type Synthetic Adsorbent; Supelco) or other suitable non-polar adsorbent or reversed-phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components. The steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e. g., 0%→100% methanol). The levels of steviol glycosides, glycosylated ent-kaurenol, and/or glycosylated ent-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization. For example, steviol glycosides can be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.
In one embodiment, a recombinant host cell capable of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture comprises a recombinant gene encoding a polypeptide capable of debranching glycogen; and/or a recombinant gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, wherein the polypeptide capable of debranching glycogen is capable of 4-α-glucanotransferase activity and α-1,6-amyloglucosidase activity, wherein the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing uridine 5′-triphosphate (UTP) from uridine diphosphate (UDP); a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate; and/or a gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1-phosphate, wherein: the polypeptide capable of debranching glycogen comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157; the polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:123; the polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:2, 119, or 143 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:141, 145, or 147; and/or the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:121 or 127, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:125, 129, 133, 135, 137, or 139 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:131.
In another embodiment, the recombinant host cell discussed above further comprises a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside; and further comprises a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding an a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid, wherein the polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:20, 22, 24, 26, 28, 30, 32, or 116; the polypeptide capable of synthesizing ent-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:34, 36, 38, 40, 42, or 120; the polypeptide capable of synthesizing ent-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:44, 46, 48, 50, or 52; the polypeptide capable of synthesizing ent-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 117, SEQ ID NO:66, 68, 70, 72, 74, or 76; the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, 92; and/or the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101, 102, 103, 104, 106, 108, 110, 112, or 114.
In another embodiment, the recombinant host cell discussed above comprises a gene encoding a polypeptide capable of debranching glycogen having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157; a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; a gene encoding a polypeptide capable of synthesizing uridine 5′-triphosphate (UTP) from uridine diphosphate (UDP) having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:123; a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having at least 60% sequence identity to the amino acid sequences set forth in any one of SEQ ID NOs:2 or 119; and a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:121; and one or more of: a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; a gene encoding a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
In another embodiment, the recombinant host cell discussed above comprises a gene encoding a polypeptide capable of debranching glycogen having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157; and/or a gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; wherein the gene encoding a polypeptide capable of debranching glycogen and/or the gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen are overexpressed relative to a corresponding host cell lacking the one or more recombinant genes, wherein the gene encoding a polypeptide capable of debranching glycogen and/or the gene encoding a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen are overexpressed by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes.
In another embodiment, the expression of the one or more recombinant genes comprising the recombinant host cell increase the amount of UDP-glucose accumulated by the recombinant host cell relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes increases the amount of UDP-glucose accumulated by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250% relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes increases an amount of the one or more steviol glycosides or the steviol glycoside composition produced by the cell relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes increases the amount of the one or more steviol glycosides produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes increases an amount of RebA, RebD, and/or RebM produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes decreases the amount of the one of one or more steviol glycosides or the steviol glycoside composition accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes decreases the amount of the one or more steviol glycosides accumulated by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell lacking the one or more recombinant genes relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes decreases an amount of 13-SMG accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes, wherein expression of the one or more recombinant genes increases the amount of total steviol glycosides produced by the cell by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes, and/or wherein expression of the one or more recombinant genes decreases the amount of total steviol glycosides produced by the cell by less than 10%, or less than 5%, or less than 2.5% relative to a corresponding host lacking the one or more recombinant genes.
In one embodiment of the recombinant host cells discussed above, the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-Stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.
In one embodiment of the recombinant host cells discussed above, the recombinant host cell is a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell or a bacterial cell.
In one embodiment, a method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture, comprises culturing the recombinant host cells discussed above in the cell culture, under conditions in which the genes are expressed, and wherein the one or more steviol glycosides or the steviol glycoside composition is produced by the recombinant host cells, wherein the genes are constitutively expressed or wherein the expression of the genes is induced, wherein the amount of RebA, RebD, and/or RebM produced by the cell is increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes, wherein the amount of 13-SMG accumulated by the cell is decreased by at least 10%, at least 25%, or at least 50% relative to a corresponding host lacking the one or more recombinant genes, wherein the amount of total steviol glycosides produced by the cell is increased by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host lacking the one or more recombinant genes, wherein the amount of total steviol glycosides produced by the cell decreases the amount of total steviol glycosides produced by the cell by less than 10%, or less than 5%, or less than 2.5% relative to a corresponding host lacking the one or more recombinant genes, wherein the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the one or more steviol glycosides or the steviol glycoside composition, and/or wherein the amount of UDP-glucose accumulated by the cell by at least 10%, at least 25%, or at least 50%, at least 100%, at least 150%, at least 200%, or at least 250% relative to a corresponding host lacking the one or more recombinant genes.
In one embodiment, the method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture further comprises isolating the produced one or more steviol glycosides or the steviol glycoside composition from the cell culture, wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or the steviol glycoside composition, and contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or the steviol glycoside composition; or contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or the steviol glycoside composition; or crystallizing or extracting the produced one or more steviol glycosides or the steviol glycoside composition; thereby isolating the produced one or more steviol glycosides or the steviol glycoside composition.
In one embodiment, the method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture further comprises recovering the one or more steviol glycosides or the steviol glycoside composition from the cell culture, wherein the produced one or more steviol glycosides or the steviol glycoside composition is enriched for the one or more steviol glycosides relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.
In one embodiment, the method of producing one or more steviol glycosides or a steviol glycoside composition comprises whole-cell bioconversion of a plant-derived or synthetic steviol and/or steviol glycosides in a cell culture of a recombinant host cell using a polypeptide capable of debranching glycogen, comprising a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157; and/or a polypeptide capable of synthesizing glucose-1-phosphate from phosphate and glycogen, comprising a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159; and optionally, one or more of a polypeptide capable of synthesizing UTP from UDP, comprising a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:123; a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, comprising a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:2, 119, or 143; or at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:141, 145, or 147; and/or a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate, comprising a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:121 or 127; at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:125, 129, 133, 135, 137, or 139; or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:131, and one or more of a polypeptide capable of glycosylating a steviol or the steviol glycoside at its C-13 hydroxyl group thereof; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof; and/or a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides or the steviol glycoside composition thereby, wherein the polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
In one embodiment, the recombinant host cell used in the method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture is a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell, wherein the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.
As used herein, the terms “or” and “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” In some embodiments, “and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, “and/or” is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through one or more of the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides (“domain swapping”). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term “functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
Typically, polypeptides that exhibit at least 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
For example, polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
Methods to modify the substrate specificity of, for example, a UGT, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., 2009, Phytochemistry 70: 325-347.
A candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120% of the length of the reference sequence, or any range between. A % identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res. 31(13):3497-500.
Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: % age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site at http://www.ebi.ac.uk/Tools/msa/clustalo/.
To determine a % identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the % identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
It will be appreciated that functional UGT proteins (e.g., a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-19 carboxyl group) can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, UGT proteins are fusion proteins. The terms “chimera,” “fusion polypeptide,” “fusion protein,” “fusion enzyme,” “fusion construct,” “chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and “chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
In some embodiments, a nucleic acid sequence encoding a UGT polypeptide (e.g., a polypeptide capable of glycosylating the steviol or the steviol glycoside at its C-19 carboxyl group) can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, Conn.). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term “domain swapping” is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a UGT polypeptide (e.g., a polypeptide capable of glycosylating a steviol or a steviol glycoside at its C-19 carboxyl group) is altered by domain swapping.
In some embodiments, a fusion protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein. In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. “Regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of steviol and/or steviol glycoside production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a steviol biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for a steviol or steviol glycoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards a steviol or steviol glycoside biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase the steviol or the steviol glycoside production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
Recombinant hosts can be used to express polypeptides for producing steviol glycosides, including, but not limited to, a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, ent-kaurene and ent-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
In some aspects, the recombinant microorganism is grown in a deep well plate. It will be understood that while data on production of steviol glycosides by the recombinant microorganism grown in deep well cultures, in some aspects, may be more easily collected than that in fermentation cultures, the small culture volume of the deep well (e.g., 1 ml or 0.5 ml) can effect differences in the environment of the microorganism and, therefore its efficiency and effectiveness in producing steviol glycosides. For example, nutrient availability, cellular waste product buildup, pH, temperature, agitation, and aeration may differ significantly between fermentation and deep well cultures. Accordingly, uptake of nutrients or other enzyme substrates may vary, affecting the cellular metabolism (e.g., changing the amount and/or profile of products accumulated by a recombinant microorganism). See, e.g., Duetz, Trends Microbiol 15(10):469-75 (2007).
Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the steviol glycosides. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate steviol and/or steviol glycosides.
Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. However, it will be appreciated that other species can be suitable to express polypeptides for the producing steviol glycosides.
For example, suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia (formally known as Hansuela), Scheffersomyces, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Humicola, lssatchenkia, Brettanomyces, Yamadazyma, Lachancea, Zygosaccharomyces, Komagataella, Kazachstania, Xanthophyllomyces, Geotrichum, Blakeslea, Dunaliella, Haematococcus, Chlorella, Undaria, Sargassum, Laminaria, Scenedesmus, Pachysolen, Trichosporon, Acremonium, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Pachysolen, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasmsonia, Thermoascus, Thielavia, Tolypocladium, Kloeckera, Pachysolen, Schwanniomyces, Trametes, Trichoderma, Acinetobacter, Nocardia, Xanthobacter, Streptomyces, Erwinia, Klebsiella, Serratia, Pseudomonas, Salmonella, Choroflexus, Chloronema, Chlorobium, Pelodictyon, Chromatium, Rhode-spirillum, Rhodobacter, Rhodomicrobium, or Yarrowia.
Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Pichia kudriavzevii, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, lssatchenkia orientalis, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Hansuela polymorpha, Brettanomyces anomalus, Yamadazyma philogaea, Fusarium fujikuroil Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida krusei, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonfi (formerly known as Talaromyces emersonfi), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla, Candida albicans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillius licheniformis, Bacillus puntis, Bacillius megaterium, Bacillius halofurans, Baciilius punilus, Serratia marcessans, Pseudomonas aeruginosa, Salmonella typhimurium, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis, Salmonella typhi, Choroflexus aurantiacus, Chloronema gigateum, Chlorobium limicola, Pelodictyon luteolum, Chromatium okenii, Rhode-spirillum rubrum, Rhodobacter spaeroides, Rhodobacter capsulatus, Rhodomicrobium vanellii, Pachysolen tannophilus, Trichosporon beigelii, and Yarrowia lipolytica.
In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Cornebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
In some embodiments, a microorganism can be a fungi from the genera including but not limited to Acremonium, Arxula, Agaricus, Aspergillus, Agaricus, Aureobasidium, Brettanomyces, Candida, Cryptococcus, Corynascus, Chrysosporium, Debaromyces, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Schizosaccharomyces, Sordaria, Scheffersomyces, Talaromyces, Rhodotorula, Rhodosporidium, Rasmsonia, Zygosaccharomyces, Thermoascus, Thielavia, Trichosporon, Tolypocladium, Trametes, and Trichoderma. Fungal species include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla.
In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Geotrichum Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, Yamadazyma philogaea, Lachancea kluyveri, Kodamaea ohmeri, or S. cerevisiae.
Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes. Thus, modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42° C., above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011, Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41:312-7).
Schizosaccharomyces is a genus of fission yeasts. Similar to S. cerevisiae, Schizosaccharomyces is a model organism in the study of eukaryotic cell biology. It provides an evolutionary distant comparison to S. cerevisiae. Species include but are not limited to S. cryophilius and S. pombe. (See Hoffman et al., 2015, Genetics. 201(2):403-23).
Humicola is a genus of filamentous fungi. Species include but are not limited to H. alopallonella and H. siamensis.
Brettanomyces is a non-spore forming genus of yeast. It is from the Saccharomycetaceae family and commonly used in the brewing and wine industries. Brettanomyces produces several sensory compounds that contribute to the complexity of wine, specifically red wine. Brettanomyces species include but are not limited to B. bruxellensis and B. claussenii. See, e.g., Fugelsang et al., 1997, Wine Microbiology.
Trichosporon is a genus of the fungi family. Trichosporon species are yeast commonly isolated from the soil, but can also be found in the skin microbiota of humans and animals. Species include, for example but are not limited to, T. aquatile, T. beigelii, and T. dermatis.
Debaromyces is a genus of the ascomycetous yeast family, in which species are characterized as a salt-tolerant marine species. Species include but are not limited to D. hansenii and D. hansenius.
Physcomitrella spp.
Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
Saccharomyces spp.
Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. Examples of Saccharomyces species include S. castellii, also known as Naumovozyma castelli.
Zygosaccharomyces spp.
Zygosaccharomyces is a genus of yeast. Originally classified under the Saccharomyces genus it has since been reclassified. It is widely known in the food industry because several species are extremely resistant to commercially used food preservation techniques. Species include but are not limited to Z. bisporus and Z. cidri. (See Barnett et al, Yeasts: Characteristics and Identification, 1983).
Geotrichum is a fungi commonly found in soil, water and sewage worldwide. It's often identified in plants, cereal and dairy products. Species include, for example but are not limited to, G. candidum and G. klebahnii (see Carmichael et al., Mycologica, 1957, 49(6):820-830.)
Kazachstania is a yeast genus in the family Sacchromycetaceae.
Torulaspora is a genus of yeasts and species include but are not limited to T. franciscae and T. globosa.
Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g., alkanes, fatty acids, and oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorganism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91(6):692-6; Bankar et al., 2009, Appl Microbiol Biotechnol. 84(5):847-65.
Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid-production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al., 2011, Applied Microbiology and Biotechnology 90(4):1219-27).
Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also, Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Candida krusei , scientific name Issatchenkia orientalis, is widely used in chocolate production. C. krusei is used to remove the bitter taste of and break down cacao beans. In addition to this species involvement in chocolate production, C. krusei is commonly found in the immunocompromised as a fungal nosocomial pathogen (see Mastromarino et al., New Microbiolgica, 36:229-238; 2013)
Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It is also commonly referred to as Komagataella pastoris. It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et al., 2014, N Biotechnol. 31(6):532-7.
Scheffersomyces stipitis also known as Pichia stipitis is a homothallic yeast found in haploid form. Commonly used instead of S. cerevisiae due to its enhanced respiratory capacity that results from and alternative respiratory system (see Papini et al., Microbial Cell Factories, 11:136 (2012)).
In some embodiments, a microorganism can be an insect cell such as Drosophilia, specifically, Drosophilia melanogaster.
In some embodiments, a microorganism can be an algal cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp.,
In some embodiments, a microorganism can be a cyanobacterial cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis.
In some embodiments, a microorganism can be a bacterial cell. Examples of bacteria include, but are not limited to, the genera Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium, and S. typhi). Bacterial cells may also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).
E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
It can be appreciated that the recombinant host cell disclosed herein can comprise a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus; a yeast cell from Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, and S. carlsbergensis), Schizosaccharomyces (e.g., S. pombe), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. glabrata, C. albicans, C. krusei, C. revkaufi, C. pulcherrima, Candida tropicalis, C. utilis, and C. boidinii), Ashbya (e.g., A. gossypii), Cyberlindnera (e.g., C. jadinii), Pichia (e.g., P. pastoris and P. kudriavzevii), Kluyveromyces (e.g., K. lactis), Hansenual (e.g., H. polymorpha), Arxula (e.g., A. adeninivorans), Xanthophyllomyces (e.g., X. dendrorhous), Issatchenkia (e.g., I. orientali), Torulaspora (e.g., T. franciscae and T. globosa), Geotrichum (e.g., G. candidum and G. klebahni), Zygosaccharomyces (e.g., Z. bisporus and Z. cidri), Yamadazyma (e.g., Y. philogaea), Lanchancea (e.g., L. kluyven), Kodamaea (e.g., K. ohmen), Brettanomyces (e.g., B. anomalus), Trichosporon (e.g., T. aquatile, T. beigelii, and T. dermatis), Debaromyces (e.g., D. hansenuis and D. hansenii), Scheffersomyces (e.g., S. stipis), Rhodosporidium (e.g., R. toruloides), Pachysolen (e.g., P. tannophilus), and Physcomitrella, Rhodotorula, Kazachstania, Gibberella, Agaricus, and Phanerochaete genera; an insect cell including, but not limited to, Drosophilia melanogaster, an algal cell including, but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis species; or a bacterial cell from Bacillus genus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, and B. pumilus) Acinetobacter, Nocardia, Xanthobacter genera, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium and S. typhi), and further including, Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum)), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides and R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii).
Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice. Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile. As used herein, the term “enriched” is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a Stevia plant. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch. In some embodiments, hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts. Thus, steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
The amount of an individual steviol glycoside (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1,500 mg/L, or about 200 to about 1,000 mg/L, at least 1,000 mg/L, at least 1,200 mg/L, at least at least 1,400 mg/L, at least 1,600 mg/L, at least 1,800 mg/L, at least 2,800 mg/L, or at least 7,000 mg/L. In some aspects, the amount of an individual steviol glycoside can exceed 7,000 mg/L. The amount of a combination of steviol glycosides (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1,500, at least 2,000 mg/L, at least 3,000 mg/L, at least 4,000 mg/L, at least 5,000 mg/L, at least 6,000 mg/L, or at least 7,000 mg/L. In some aspects, the amount of a combination of steviol glycosides can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
The amount of compounds accumulated by the recombinant host may be reported as a “flux.” For example, the “total flux” may be calculated as a sum (in g/L RebD equivalents) of measured RebA, RebB, RebD, RebE, RebM, 13-SMG, rubusoside, steviol-1,2-bioside, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, copalol, ent-kaurenoic acid, glycosylated ent-kaurenoic acid, glycosylated ent-kaurenol, ent-kaurenal, geranylgeraniol, ent-kaurenal, and ent-kaurene levels. Individual compounds, such as individual steviol glycosides, or groups of compounds, such as the group of steviol glycosides, may be reported as a fraction of total flux. For example, “steviol glycoside/flux” may calculated as ((“total flux”−(geranylgeraniol+copalol+ent-kaurene+glycosylated ent-kaurenol+ent-kaurenol+ent-kaurenal+ent-kaurenoic acid+glycosylated ent-kaurenoic acid)/“total flux”).
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce steviol and/or steviol glycosides. For example, a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor, while a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions. See, e.g., WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
For example, substantially pure steviol or steviol glycoside such as RebM or RebD can be included in food products such as ice cream, carbonated drinks, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces. Substantially pure steviol or the steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements. Substantially pure steviol or the steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry. Alternatively, a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol glycoside, recovering the steviol or the steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
In another alternative, a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g., saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium. The weight ratio of the steviol or the steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product. See, e.g., U.S. 2007/0128311. In some embodiments, the steviol or the steviol glycoside may be provided with a flavor (e.g., citrus) as a flavor modulator.
Compositions produced by a recombinant microorganism described herein can be incorporated into food products. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g., ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g., chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g., fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. In some embodiments, a steviol glycoside composition produced herein is a component of a pharmaceutical composition. See, e.g., Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.; EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), “Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive,” 2010, EFSA Journal 8(4):1537; U.S. Food and Drug Administration GRAS Notice 323; U.S Food and Drug Administration GRAS Notice 329; WO 2011/037959; WO 2010/146463; WO 2011/046423; and WO 2011/056834.
For example, such a steviol glycoside composition can have from 90-99 weight % RebA and an undetectable amount of Stevia plant-derived contaminants, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebB-enriched composition has an undetectable amount of Stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebD-enriched composition has an undetectable amount of Stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebE-enriched composition has an undetectable amount of Stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebM-enriched composition has an undetectable amount of Stevia plant-derived contaminants.
In some embodiments, a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or “cup-for-cup” product. Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art. Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM, can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use. In some embodiments, a steviol glycoside produced in vitro, in vivo, or by whole cell bioconversion.
The invention also provides an isolated nucleic acid molecule encoding a polypeptide or a catalytically active portion thereof capable of debranching glycogen comprising a polypeptide or a catalytically active portion thereof having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157 or a polypeptide or a catalytically active portion thereof capable of synthesizing glucose-1-phosphate comprising a polypeptide or a catalytically active portion thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159.
In one aspect of the isolated nucleic acids disclosed herein, the nucleic acid is cDNA.
The invention also provides a polypeptide or a catalytically active portion thereof capable of debranching glycogen comprising a polypeptide or a catalytically active portion thereof having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:157 or a polypeptide or a catalytically active portion thereof capable of synthesizing glucose-1-phosphate comprising a polypeptide or a catalytically active portion thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:159.
In one aspect of the polypeptides or the catalytically active portion thereof disclosed herein, the polypeptide or the catalytically active portion thereof is a purified polypeptide or a catalytically active portion thereof.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Steviol glycoside-producing S. cerevisiae strains were constructed as described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in its entirety. For example, yeast strains comprising and expressing a native gene encoding a YNK1 polypeptide (SEQ ID NO:122, SEQ ID NO:123), a native gene encoding a PGM1 polypeptide (SEQ ID NO:1, SEQ ID NO:2), a native gene encoding a PGM2 polypeptide (SEQ ID NO:118, SEQ ID NO:119), a native gene encoding a UGP1 polypeptide (SEQ ID NO:120, SEQ ID NO:121), a native gene encoding a GDB1 polypeptide (SEQ ID NO:156, SEQ ID NO:157), a native gene encoding a GPH1 polypeptide (SEQ ID NO:158, SEQ ID NO:159), a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO:19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide (SEQ ID NO:51, SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding a KO polypeptide (SEQ ID NO:63, SEQ ID NO:64), a recombinant gene encoding an ATR2 polypeptide (SEQ ID NO:91, SEQ ID NO:92), a recombinant gene encoding a KAHe1 polypeptide (SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding a CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding a CPR1 polypeptide (SEQ ID NO:77, SEQ ID NO:78), a recombinant gene encoding a UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID NO:9), a recombinant gene encoding a UGT85C2 polypeptide (SEQ ID NO:5/SEQ ID NO:6, SEQ ID NO:7), a recombinant gene encoding a UGT74G1 polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a UGT91d2e-b polypeptide (SEQ ID NO:12, SEQ ID NO:13), a recombinant gene encoding an EUGT11 polypeptide (SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16), a recombinant gene encoding a KAH polypeptide (SEQ ID NO:96, SEQ ID NO:97), a recombinant gene encoding a KO polypeptide (SEQ ID NO:117, SEQ ID NO:64), and additional copies of the gene encoding a YNK1 polypeptide (SEQ ID NO:122, SEQ ID NO:123), the gene encoding a PGM1 polypeptide (SEQ ID NO:1, SEQ ID NO:2), the gene encoding a PGM2 polypeptide (SEQ ID NO:118, SEQ ID NO:119), the gene encoding a UGP1 polypeptide (SEQ ID NO:120, SEQ ID NO:121), and the gene encoding an ERC1 transporter polypeptide (i.e., of the MATE family) (SEQ ID NO:160, SEQ ID NO:161) were engineered to accumulate steviol glycosides.
A steviol glycoside-producing S. cerevisiae strain as described in Example 1 was transformed with vectors comprising additional copies of the gene encoding a GDB1 polypeptide (SEQ ID NO:156, SEQ ID NO:157), operably linked to a TPI1 promoter (SEQ ID NO:152) and a ADH1 terminator (SEQ ID NO:155) and the gene encoding a GPH1 polypeptide (SEQ ID NO:158, SEQ ID NO:159), operably linked to a pPDC1 promoter (SEQ ID NO:153) and a tCYC1 terminator (SEQ ID NO:154).
Fed-batch fermentation with cultures of the transformed S. cerevisiae strain and a control S. cerevisiae strain (a steviol glycoside-producing S. cerevisiae strain as described in Example 1) was carried out aerobically in 2L fermenters at 30° C. with an approximate 16 h growth phase in minimal medium comprising glucose, ammonium sulfate, trace metals, vitamins, salts, and buffer followed by an approximate 100 h feeding phase with a glucose-comprising defined feed medium. A pH near 6.0 and glucose-limiting conditions were maintained. Extractions of whole culture samples (without cell removal) were performed and extracts were analyzed by LC-UV to determine levels of steviol glycosides.
LC-UV was conducted with an Agilent 1290 instrument comprising a variable wavelength detector (VWD), a thermostatted column compartment (TCC), an autosampler, an autosampler cooling unit, and a binary pump, using SB-C18 rapid resolution high definition (RRHD) 2.1 mm×300 mm, 1.8 μm analytical columns (two 150 mm columns in series; column temperature of 65° C.). Steviol glycosides were separated by a reversed-phase C18 column followed by detection by UV absorbance at 210 mm. Quantification of steviol glycosides was done by comparing the peak area of each analyte to standards of RebA and applying a correction factor for species with differing molar absorptivities. For LC-UV, 0.5 mL cultures were spun down, the supernatant was removed, and the wet weight of the pellets was calculated. The LC-UV results were normalized by pellet wet weight. Total steviol glycoside values of the fed-batch fermentation were calculated based upon the measured levels of steviol glycosides calculated as a sum (in g/L RebD equivalents) of measured RebA, RebB, RebD, RebE, RebM, 13-SMG, rubusoside, steviol-1,2-bioside, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, and hepta-glycosylated steviol. Total flux was calculated as a sum (in g/L RebD equivalents) of measured RebA, RebB, RebD, RebE, RebM, 13-SMG, rubusoside, steviol-1,2-bioside, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, copalol, ent-kaurenoic acid, glycosylated ent-kaurenoic acid, glycosylated ent-kaurenol, ent-kaurenal, geranylgeraniol, ent-kaurenal, and ent-kaurene levels. Results are shown in Table 1.
Percent change in steviol glycoside production (% increase or % decrease) was calculated as follows. The amount of a particular steviol glycoside (e.g., RebM) produced by the control strain (in g/L) was subtracted from the amount of the particular steviol glycoside produced by the experimental strain overexpressing GPH1 and GDB1 (in g/L). That resulting value was then divided by the amount of the particular steviol glycoside produced by the control strain (in g/L) and multiplied by 100. A positive number using this equation signifies a percent increase in a particular steviol glycoside produced by the strain overexpressing GPH1 and GDB1 (in g/L), whereas a negative number using this equation signifies a percent decrease in a particular steviol glycoside (e.g., 13-SMG) produced by the strain overexpressing GPH1 and GDB1 (in g/L).
Overexpression of GPH1 and GDB1 resulted in a 29% decrease in 13-SMG accumulation, and an increase of 8%, 29% and 12% in RebA, RebD and RebM accumulation, respectively, in comparison to the control strain. There was also a 14% increase in RebD+RebM accumulation. Furthermore, there was a 14% increase in total flux accumulated by the strain overexpressing GPH1 and GDB1 genes, compared to the control strain. The total amount of steviol glycosides accumulated changed negligibly. Without being bound by theory, the lack of a significant change in total steviol glycoside accumulation and the decrease in 13-SMG accumulation suggests that overexpression of GPH1 and GDB1 in a steviol glycoside producing recombinant host enhances the flux of glycosylation pathways towards higher molecular weight steviol glycosides, e.g. RebD and RebM, altering the production profile, rather than simply increasing steviol glycoside production, generally.
Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
S. cerevisiae
S. cerevisiae
S. rebaudiana
S. rebaudiana
S. rebaudiana
S. rebaudiana
S. rebaudiana
S. rebaudiana
O. sativa
O. sativa
S. rebaudiana
G. fujikuroi
M. musculus
S. clavuligerus
S. acidocaldarius
Synechococcus sp.
S. rebaudiana
S. clavuligerus
B. japonicum
Z. mays
A. thaliana
S. rebaudiana
S. rebaudiana
Z. mays
P. trichocarpa
A. thaliana
P. amygdali
P. patens
G. fujikuroi
S. rebaudiana
L. sativa
R. suavissimus
C. mollissima
T. halophila
V. vinifera
G. fujikuroi
T. versicolor
A. thaliana
S. rebaudiana
S. grosvenorii
S. grosvenorii
G. fujikuroi
S. rebaudiana
S. rebaudiana
S. rebaudiana
A. thaliana
A. thaliana
S. rebaudiana
R. suavissimus
R. suavissimus
P. avium
P. avium
P. mume
P. mume
P. mume
P. persica
S. rebaudiana
S. rebaudiana
A. thaliana
V. vinifera
M. truncatula
A. thaliana
R. suavissimus
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. rebaudiana
S. rebaudiana
A. pullulans
A. pullulans
A. thaliana
A. thaliana
E. coli
E. coli
R. suavissimus
R. suavissimus
H. vulgare
H. vulgare
O. sativa
O. sativa
S. tuberosum
S. tuberosum
A. thaliana
A. thaliana
E. coli
E. coli
R. suavissimus
R. suavissimus
S. rebaudiana
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/083689 | 12/5/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62594612 | Dec 2017 | US |
